Molecules and methods for iterative polypeptide analysis and processing

ABSTRACT

Reagents and methods for the digital analysis of proteins or peptides are provided. Specifically provided herein are proteins for identifying the N-terminal amino acid or N-terminal phosphorylated amino acid of a polypeptide. Also, an enzyme for use in the cleavage step of the Edman degradation reaction and a method for using this enzyme are described.

REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.14/211,448, filed Mar. 14, 2014, which claims the benefit of U.S.Provisional Application No. 61/798,705, filed Mar. 15, 2013, the entiredisclosures of which are incorporated herein by reference.

GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under grant R01 GM101602awarded by the National Institutes of Health. The Government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to reagents and methods for thedigital analysis of proteins or peptides. Specifically provided hereinare proteins for identifying the N-terminal amino acid or N-terminalphosphorylated amino acid of a polypeptide. Another aspect of theinvention is an enzyme for use in the cleavage step of the Edmandegradation reaction and a method for using this enzyme.

BACKGROUND OF THE INVENTION

Proteins carry out the majority of signaling, metabolic, and regulatorytasks necessary for life. As a result, a quantitative description of theproteomic state of cells, tissues, and fluids is crucial for assessingthe functionally relevant differences between diseased and unaffectedtissues, between cells of different lineages or developmental states,and between cells executing different regulatory programs. Althoughpowerful high-throughput techniques are available for determining theRNA content of a biological sample, the correlation between mRNA andprotein levels is low (1).

The preferred method for proteomic characterization is currently massspectrometry. Despite its many successes, mass spectrometry possesseslimitations. One limitation is quantification. Because differentproteins ionize with different efficiencies, it is difficult to comparerelative amounts between two samples without isotopic labeling (2). In‘shotgun’ strategies for analyzing complex samples, the uncertainties ofpeptide assignment further complicate quantification, especially for lowabundance proteins (3). A second limitation of mass spectrometry is itsdynamic range. For unbiased samples that have not undergoneprefractionation or affinity purification, the dynamic range in analyteconcentration is roughly 10²-10³, depending upon the instrument (4).This is problematic for complex samples such as blood, where twoproteins whose levels are measured in clinical laboratories (albumin andinterleukin-6) can differ in abundance by 10¹⁰ (5). Another limitationis the analysis of phosphopeptides, due to the loss of phosphate in someionization modes. The power of proteomic approaches would increasedramatically with the introduction of a more quantitativehigh-throughput assay possessing greater dynamic range.

One promising technology for the analysis of proteins in a sensitive andquantitative manner was developed by Mitra et al (7). This technology,referred to as Digital Analysis of Proteins by End Sequencing or DAPES,features a method for single molecule protein analysis. To performDAPES, a large number (ca. 10⁹) of protein molecules are denatured andcleaved into peptides. These peptides are immobilized on a nanogelsurface applied to the surface of a microscope slide and their aminoacid sequences are determined in parallel using a method related toEdman degradation. Phenyl isothiocyanate (PITC) is added to the slideand reacts with the N-terminal amino acid of each peptide to form astable phenylthiourea derivative. Next, the identity of the N-terminalamino acid derivative is determined by performing, for example, 20rounds of antibody binding with antibodies specific for eachPITC-derivatized N-terminal amino acid, detection, and stripping. TheN-terminal amino acid is removed by raising the temperature or loweringpH, and the cycle is repeated to sequence 12-20 amino acids from eachpeptide on the slide. The absolute concentration of every protein in theoriginal sample can then be calculated based on the number of differentpeptide sequences observed.

The phenyl isothiocyanate chemistry used in DAPES is the same used inEdman degradation and is efficient and robust (>99% efficiency).However, the cleavage of single amino acids requires strong anhydrousacid or alternatively, an aqueous buffer at elevated temperatures.Cycling between either of these harsh conditions is undesirable formultiple rounds of analysis on sensitive substrates used for singlemolecule protein detection (SMD). Thus, there is a need in the art forimproved reagents and methods for the parallel analysis of peptides insingle molecule protein detection (SMD) format.

SUMMARY OF THE INVENTION

One aspect of the invention is an improved method for single moleculesequencing of proteins or peptides. Generally, the method for sequencinga polypeptide, the method comprises (a) contacting the polypeptide withone or more fluorescently labeled N-terminal amino acid binding proteins(NAABs); (b) detecting fluorescence of a NAAB bound to an N-terminalamino acid of the polypeptide; (c) identifying the N-terminal amino acidof the polypeptide based on the fluorescence detected; (d) removing theNAAB from the polypeptide; (e) optionally repeating steps (a) through(d); (f) cleaving the N-terminal amino acid of the polypeptide via Edmandegradation; and (g) repeating steps (a) through (f) one or more times.

The present invention also generally relates to reagents for the digitalanalysis of proteins or peptides. Specifically provided herein areproteins for identifying the N-terminal amino acid or N-terminalphosphorylated amino acid of a polypeptide.

Another aspect of the invention relates to an enzyme for use in thecleavage step of the Edman degradation reaction and a method for usingthis enzyme. Generally, the enzymatic Edman degradation method comprisesreacting the N-terminal amino acid of the polypeptide with phenylisothiocyanate (PITC) to form a PITC-derivatized N-terminal amino acidand cleaving the PITC-derivatized N-terminal amino acid using an Edmandegradation enzyme.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the Digital Analysis of Proteins by End SequencingProtocol (DAPES) utilizing N-terminal amino acid binding proteins in theidentification step and a synthetic enzyme in the cleavage step.

FIGS. 2A-2B show the binding specificity of wild-type E. coli methionineaminopeptidase (eMAP) and an engineered leucine-specific aminopeptidase(eLAP) of the present invention in a single-molecule detectionexperiment.

FIG. 3 shows the binding specificity of an engineered mutant ofmethionine tRNA synthetase (MetRS) of the present invention thatexhibits binding specificity for surface-immobilized peptides withN-terminal methionines.

FIG. 4A-4B depict three mutations (indicated by the arrows) introducedinto a model of cruzain (pdb code: 1U9Q (27)) to accommodate the phenylmoiety of the Edman reagent phenyl isothiocyanate.

FIG. 5A depicts a model for a cleavage intermediate for Edmandegradation generated using experimental small molecules structures forsimilar compounds and geometrically optimized using quantum chemistrycalculations.

FIG. 5B shows the model for the intermediate fitted into the active sitecleft of the enzyme cruzain. The wild-type catalytic cysteine wasremoved. The activating residues (the other two components of the‘catalytic triad’) were retained. These are a histidine and asparaginethat are intended to activate the sulfur atom in the Edman reagent fornucleophile attack on the peptide bond.

FIG. 6 is a graphical representation of kinetic data from cleavageexperiments using an Edman degradation enzyme of the present inventionand the substrate Ed-Asp-AMC.

FIG. 7 is a trace plot of biolayer interferometry kinetics data showingthe binding affinity of two proteins for peptides with N-terminalhistidine residues: (1) engineered His NAAB (open circles); (2) nativewild-type protein (solid circles).

FIG. 8 is a full binding matrix showing the binding affinity of everysingle NAAB (row) for a single N-terminal amino acid (column) asmeasured by biolayer interferometry.

DESCRIPTION OF THE INVENTION

In one aspect, the present invention is directed to a method andreagents for sequencing a polypeptide. In particular, the presentinvention provides methods and reagents for the single-molecule,high-throughput sequencing of polypeptides. Recent advances insingle-molecule protein detection (SMD) allow for the parallel analysisof large numbers of individual proteins utilizing digital protocols. Inaccordance with the present invention, reagents capable of specificallybinding to N-terminal amino acids for an identification step areprovided.

The present invention also includes methods and reagents foridentification phosphorylated N-terminal amino acids. Quantitativelyinterrogating peptide sequences in neutral aqueous environments allowsfor the possibility of proteomic analyses complementary to thoseafforded by mass spectrometry. The N-terminal amino acids specific forphosphorylated forms of amino acids allow for quantitative comparison ofproteomic inventories and signal transduction cascades in differentsamples.

In another aspect, the present invention is directed to a method andreagents for enzymatic Edman degradation (i.e., for enzymaticallycleaving the N-terminal amino group of a polypeptide). In accordancewith this aspect, a synthetic enzyme is provided that catalyzes thecleavage step of the Edman degradation reaction in an aqueous buffer andat neutral pH, thereby providing an alternative to the harsh chemicalconditions typically employed in Edman degradation.

Yet another aspect of the present invention is directed to an integratedhigh-throughput method for sequencing of polypeptides that includes useof reagents capable of specifically binding to N-terminal amino acidsfor an identification step and use of an enzymatic Edman degradation toremove N-terminal amino acids.

I. N-Terminal Amino Acids Binders (NAABs)

In accordance with the present invention, reagents capable ofspecifically binding to N-terminal amino acids are provided. In variousaspects of the invention, the N-terminal amino acid binders (NAABs) eachselectively bind to a particular amino acid, for example one of thetwenty standard naturally occurring amino acids. The standard,naturally-occurring amino acids include Alanine (A or Ala), Cysteine (Cor Cys), Aspartic Acid (D or Asp), Glutamic Acid (E or Glu),Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His),Isoleucine (I or Ile), Lysine (K or Lys), Leucine (L or Leu), Methionine(M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q orGln), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr),Valine (V or Val), Tryptophan (W or Trp), and Tyrosine (Y or Tyr).

The NAABs of the present invention can be made by modifying variousnaturally occurring proteins to introduce one or more mutations in theamino acid sequence to produce engineered proteins that bind toparticular N-terminal amino acids. For example, aminopeptidases or tRNAsynthetases can be modified to create NAABs that selectively bind toparticular N-terminal amino acids.

A. eLAP

For example, a NAAB that binds specifically to N-terminal leucineresidues has been developed by introducing mutations into E. colimethionine aminopeptidase (eMAP). This NAAB (eLAP) has 19 amino acidsubstitutions as compared to wild-type eMAP. In particular, eLAP hassubstitutions at the amino acid positions corresponding to positions 42,46, 56-60, 62, 63, 65-70, 81, 101, 177, and 221 of wild-type eMAP. IneLAP, the aspartate at position 42 of eMAP is replaced with a glutamate,the asparagine at position 46 of eMAP is replaced with a tryptophan, thevaline at position 56 of eMAP is replaced with a threonine, the serineat position 57 of eMAP is replaced with an aspartate, the alanine atposition 58 of eMAP is replaced with a serine, the cysteine at position59 of eMAP is replaced with a leucine, the leucine at position 60 ofeMAP is replaced with a threonine, the tyrosine at position 62 of eMAPis replaced with a histidine, the histidine at position 63 of eMAP isreplaced with an asparagine, the tyrosine at position 65 of eMAP isreplaced with a isoleucine, the proline at position 66 of eMAP isreplaced with an aspartate, the lysine at position 67 of eMAP isreplaced with a glycine, the serine at position 68 of eMAP is replacedwith a histidine, the valine at position 69 of eMAP is replaced with aglycine, the cysteine at position 70 of eMAP is replaced with a serine,the isoleucine at position 81 of eMAP is replaced with a valine, theisoleucine at position 101 of eMAP is replaced with an arginine, thephenylalanine at position 177 of eMAP is replaced with a histidine, andthe tryptophan at position 221 of eMAP is replaced with a serine.Alternative substitutions could be made at selected positions. Forexample, valine at 56 could be replaced instead by serine, leucine at 60could be replaced instead by serine, tyrosine at 65 could be replacedinstead by valine, cysteine at 70 could be replaced instead bythreonine, and tryptophan at 221 could be replaced instead by threonine.

Accordingly, one reagent in accordance with the present inventioncomprises an isolated, synthetic, or recombinant NAAB comprising anamino acid sequence having a glutamate residue at a positioncorresponding to position 42 of wild-type E. coli methionineaminopeptidase (eMAP) (SEQ ID NO: 1), a tryptophan residue at a positioncorresponding to position 46 of wild-type eMAP, a threonine or serineresidue at a position corresponding to position 56 of wild-type eMAP, anaspartate residue at a position corresponding to position 57 ofwild-type eMAP, a serine residue at a position corresponding to position58 of wild-type eMAP, a leucine residue at a position corresponding toposition 59 of wild-type eMAP, a threonine or serine residue at aposition corresponding to position 60 of wild-type eMAP, a histidineresidue at a position corresponding to position 62 of wild-type eMAP, anasparagine residue at a position corresponding to position 63 ofwild-type eMAP, a isoleucine or valine residue at a positioncorresponding to position 65 of wild-type eMAP, an aspartate residue ata position corresponding to position 66 of wild-type eMAP, a glycineresidue at a position corresponding to position 67 of wild-type eMAP, ahistidine residue at a position corresponding to position 68 ofwild-type eMAP, a glycine residue at a position corresponding toposition 69 of wild-type eMAP, a serine or threonine residue at aposition corresponding to position 70 of wild-type eMAP, a valineresidue at a position corresponding to position 81 of wild-type eMAP, anarginine residue at a position corresponding to position 101 ofwild-type eMAP, a histidine residue at a position corresponding toposition 177 of wild-type eMAP, and a serine or threonine residue at aposition corresponding to position 221 of wild-type eMAP.

The remaining amino acid sequence of the NAAB comprises a sequencesimilar to that of wild-type eMAP, but which may contain additionalamino acid mutations (including deletions, insertions, and/orsubstitutions), so long as such mutations do not significantly impairthe ability of the NAAB to selectively bind to N-terminal leucineresidues. For example, the remaining amino acid sequence can comprise anamino acid sequence having at least about 80% sequence identity to theamino acid sequence of wild-type eMAP (SEQ ID NO: 1), or at least 85%,at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 1.

In some aspects of the present invention, the NAAB comprises the aminoacid sequence of SEQ ID NO: 2. For example, the NAAB can consist of theamino acid sequence of SEQ ID NO: 2.

The NAAB preferably selectively binds to N-terminal leucine residueswith at least about a 1.5:1 ratio of specific to non-specific binding,more preferably about a 2:1 ratio of specific to non-specific binding.Non-specific binding refers to background binding, and is the amount ofsignal that is produced when the amino acid target of the NAAB is notpresent at the N-terminus of an immobilized peptide.

B. tRNA Synthetase-Based NAABs

1. N-Terminal Methionine Binding Protein

NAABs can also be made by introducing mutations into class I and classII tRNA synthetases (RSs). NAABs for use in the polypeptide sequencingprocesses described herein should possess high affinity and specificityfor amino acids at the N-terminus of peptides. Because tRNA synthetaseshave intrinsic specificity for free amino acids, they are usefulscaffolds for developing NAABs for use in protein sequencing. Theinherent specificity of these scaffold proteins is retained, whilebroadening the binding capabilities of these proteins from free monomersto peptides, and removing unnecessary domains or functions. The ProteinData Bank contains multiple crystal structures for RSs specific for alltwenty canonical amino acids. Moreover, unlike other classes of aminoacid binding molecules, such as riboswitches, RSs do not envelop theentire amino acid, as the C-terminus must be available for adenylation.The binding pocket in these molecules can be modified to permit theentry of peptides presenting the specifically bound amino acid. Thisresults in a complete set of engineered RS fragments that can bind totheir cognate amino acids at the N-termini of peptides.

The class I RS proteins form a distinct structural family that isidentified by sequence homology and has been extensively characterizedboth biochemically and biophysically. RS proteins possess a modulararchitecture, and the domains conferring specificity for a particularamino acid are readily identified (18). Several types of mutations toimprove the performance of the amino acid binding domain of an RS as aNAAB can be introduced. First, one or more mutations can be introducedinto the binding domain to lock the domain into the bound conformation,eliminating the energetic cost of any induced conformational change(16). Second, one or more mutations can be introduced to widen thebinding pocket for the amino acid, making room for entry of a peptide.This approach can be used for each of the RS proteins.

For example, mutations can be introduced into methionyl-tRNA synthetase(MetRS) from E. coli to create a NAAB that binds specifically toN-terminal methionine residues. This NAAB comprises a truncated versionof wild-type E. coli MetRS (residues 4-547; SEQ ID NO: 3) having foursubstitution mutations as compared to the wild-type sequence (SEQ ID NO:5). The sequence of this N-terminal methionine-specific NAAB is providedby SEQ ID NO: 4. In particular, in the methionine-specific NAAB, theleucine at position 13 of wild-type E. coli MetRS is replaced with aserine (L13S), the phenylalanine at position 260 is replaced with aleucine (Y260L), the aspartic acid at position 296 is replaced with aglycine (D296G), and the histidine at position 301 is replaced with aleucine (H301L).

Accordingly, one reagent in accordance with the present inventioncomprises an isolated, synthetic, or recombinant NAAB comprising anamino acid sequence having a serine residue at a position correspondingto position 13 of wild-type E. coli methionyl-tRNA synthetase (MetRS); aleucine residue at a position corresponding to position 260 of wild-typeE. coli MetRS; a glycine residue at a position corresponding to position296 of wild-type E. coli MetRS; and a leucine residue at a positioncorresponding to position 301 of wild-type E. coli MetRS.

The remaining amino acid sequence of the NAAB comprises a sequencesimilar to that of amino acids 4-547 of wild-type MetRS, but may containadditional amino acid mutations (including deletions, insertions, and/orsubstitutions), so long as such mutations do not significantly impairthe ability of the NAAB to selectively bind to N-terminal methionineresidues. For example, the remaining amino acid sequence can comprise anamino acid sequence having at least about 80% sequence identity to theamino acid sequence of SEQ ID NO: 3, or at least 85%, at least 90%, atleast 93%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the amino acid sequence of SEQID NO: 3.

In certain aspects of the invention, the NAAB comprises the amino acidsequence of SEQ ID NO: 4. For example, the NAAB can consist of the aminoacid sequence of SEQ ID NO: 4.

The NAAB preferably selectively binds to N-terminal methionine residueswith at least about a 2:1 ratio of specific to non-specific binding,more preferably at least about a 7:1 ratio, at least about a 10:1 ratio,or about a 13:1 ratio of specific to non-specific binding.

2. N-Terminal Phenylalanine Binding Protein

The starting point for the phenylalanine NAAB (Phe NAAB) was thephenylalanine-tRNA synthetase (PheRS) from Thermus Thermophilus, forwhich a crystal structure is available. Normally the operational unit isa tetramer with two copies each of two separate proteins. Only one ofthe proteins has the amino acid binding specificity, so a model was madeof one copy of the protein in isolation. The N-terminus of the proteinwas truncated, which exposed a significant amount of surface area thatwas previously buried in contacts with other proteins. This surface washydrophobic, and mutations were made the surface to make the proteinstabile and soluble as a monomer. Tighter binding of the mutant topeptides was observed when compared to the wild-type protein.

For example, mutations can be introduced into PheRS from ThermusThermophilus to create a NAAB that binds specifically to N-terminalphenylalanine residues. This NAAB comprises a truncated version ofwild-type Thermus Thermophilus PheRS (residues 86-350; SEQ ID NO: 6)having 22 substitution mutations as compared to the wild-type sequence.The sequence of this N-terminal phenylalanine-specific NAAB is providedby SEQ ID NO: 7. In particular, PheNAAB has substitutions at the aminoacid positions corresponding to positions 100, 142, 143, 152-154, 165,205, 212, 228-232, 234, 257, 287, 289, 303, 336, 338, 340 of wild-typePheRS. In the NAAB, the leucine at position 100 of PheRS is replacedwith an aspartate, the histidine at position 142 of PheRS is replacedwith an asparagine, the histidine at position 143 of PheRS is replacedwith a glycine, the phenylalanine at position 152 of PheRS is replacedwith a valine, the tryptophan at position 153 of PheRS is replaced witha glycine, the leucine at position 154 of PheRS is replaced with alysine, the leucine at position 165 of PheRS is replaced with anaspartate, the phenylalanine at position 205 of PheRS is replaced withan alanine, the histidine at position 212 of PheRS is replaced with analanine, the isoleucine at position 228 of PheRS is replaced with avaline, the alanine at position 229 of PheRS is replaced with anasparagine, the methionine at position 230 of PheRS is replaced with aglutamate, the alanine at position 231 of PheRS is replaced with aglycine, the histidine at position 232 of PheRS is replaced with anaspartate, the lysine at position 234 of PheRS is replaced with atyrosine, the tyrosine at position 257 of PheRS is replaced with athreonine, the histidine at position 287 of PheRS is replaced with aglycine, the lysine at position 289 of PheRS is replaced with anasparagine, the leucine at position 303 of PheRS is replaced with anaspartate, the phenylalanine at position 336 of PheRS is replaced withan alanine, the glycine at position 338 of PheRS is replaced with athreonine, and the leucine at position 340 of PheRS is replaced with aglycine.

Accordingly, one reagent in accordance with the present inventioncomprises an isolated, synthetic, or recombinant NAAB comprising anamino acid sequence having a an aspartate residue at a positioncorresponding to position 100 of wild-type PheRS from ThermusThermophilus (SEQ ID NO: 8), an asparagine residue at a positioncorresponding to position 142 of wild-type PheRS, a glycine residue at aposition corresponding to position 143 of wild-type PheRS, a valineresidue at a position corresponding to position 152 of wild-type PheRS,a glycine residue at a position corresponding to position 153 ofwild-type PheRS, a lysine residue at a position corresponding toposition 154 of wild-type PheRS, an aspartate residue at a positioncorresponding to position 165 of wild-type PheRS, an alanine residue ata position corresponding to position 205 of wild-type PheRS, an alanineresidue at a position corresponding to position 212 of wild-type PheRS,a valine residue at a position corresponding to position 228 ofwild-type PheRS, an asparagine residue at a position corresponding toposition 229 of wild-type PheRS, a glutamate residue at a positioncorresponding to position 230 of wild-type PheRS, a glycine residue at aposition corresponding to position 231 of wild-type PheRS, an aspartateresidue at a position corresponding to position 232 of wild-type PheRS,a tyrosine residue at a position corresponding to position 234 ofwild-type PheRS, a threonine residue at a position corresponding toposition 257 of wild-type PheRS, a glycine residue at a positioncorresponding to position 287 of wild-type PheRS, an asparagine residueat a position corresponding to position 289 of wild-type PheRS, anaspartate residue at a position corresponding to position 303 ofwild-type PheRS, an alanine residue at a position corresponding toposition 336 of wild-type PheRS, a threonine residue at a positioncorresponding to position 338 of wild-type PheRS, and a glycine residueat a position corresponding to position 340 of wild-type PheRS.

The remaining amino acid sequence of the NAAB comprises a sequencesimilar to that of wild-type PheRS, but which may contain additionalamino acid mutations (including deletions, insertions, and/orsubstitutions), so long as such mutations do not significantly impairthe ability of the NAAB to selectively bind to N-terminal phenylalanineresidues. For example, the remaining amino acid sequence can comprise anamino acid sequence having at least about 80% sequence identity to theamino acid sequence of truncated wild-type PheRS (SEQ ID NO: 6), or atleast 85%, at least 90%, at least 93%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO:6.

In some aspects of the present invention, the NAAB comprises the aminoacid sequence of SEQ ID NO: 7. For example, the NAAB can consist of theamino acid sequence of SEQ ID NO: 7.

The NAAB preferably selectively binds to N-terminal phenylalanineresidues with at least about a 1.5:1 ratio of specific to non-specificbinding, more preferably about a 2:1 ratio of specific to non-specificbinding.

3. N-Terminal Histidine Binding Protein

The starting point for the histidine NAAB (His NAAB) was thehistidine-tRNA synthetase (HisRS) from E. coli, for which a crystalstructure is available. The fragment of wild-type HisRS from 1-320 wasshown to be monomeric by others. After inspecting the crystal structure,further residues were truncated from both ends. The initial fragmenttested has from Lysine3 to Alanine180. Protein design was conducted toreplace a long loop near the binding site with a shorter loop that wouldcreate a more open pocket and result in tighter binding to N-terminalhistidine residues. This involved the removal of 7 residues (fromArginine113 to Lysine119) and two mutations wherein the arginine atposition 121 of HisRS is replaced with an asparagine, and the tyrosineat position 122 of HisRS is replaced with an alanine. Thus, thus thisNAAB comprises a truncated version of wild-type E. coli HisRS (residues3-180; SEQ ID NO: 10) having two substitution mutations as compared tothe wild-type sequence. The sequence of this N-terminalhistidine-specific NAAB is provided by SEQ ID NO: 9.

Accordingly, one reagent in accordance with the present inventioncomprises an isolated, synthetic, or recombinant NAAB comprising anamino acid sequence having an asparagine residue at a positioncorresponding to position 121 of wild-type HisRS from E. coli (SEQ IDNO: 9) and an alanine residue at a position corresponding to position122 of wild-type HisRS.

The remaining amino acid sequence of the NAAB comprises a sequencesimilar to that of wild-type HisRS, but which may contain additionalamino acid mutations (including deletions, insertions, and/orsubstitutions), so long as such mutations do not significantly impairthe ability of the NAAB to selectively bind to N-terminal histidineresidues. For example, the remaining amino acid sequence can comprise anamino acid sequence having at least about 80% sequence identity to theamino acid sequence of wild-type HisRS (SEQ ID NO: 9), or at least 85%,at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 9.

In some aspects of the present invention, the NAAB comprises the aminoacid sequence of SEQ ID NO: 10. For example, the NAAB can consist of theamino acid sequence of SEQ ID NO: 10.

The NAAB preferably selectively binds to N-terminal histidine residueswith at least about a 1.5:1 ratio of specific to non-specific binding,more preferably about a 2:1 ratio of specific to non-specific binding.

4. Other NAABs

Full-length or truncated fragments from wild-type synthetases from E.coli may be used as NAABs for the remaining amino acids. See Table A forthe sequences of each of the NAABs. Accordingly, in some aspects of thepresent invention, the NAAB comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO:13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ IDNO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27;and SEQ ID NO: 28. In various embodiments, a set of NAABs comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of theamino acid sequences of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 7; SEQ IDNO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19;SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO:24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; and SEQ ID NO: 28. Forexample, a set of NAABs comprises of the amino acid sequences of SEQ IDNO: 2; SEQ ID NO: 4; SEQ ID NO: 7; SEQ ID NO: 10; SEQ ID NO: 11; SEQ IDNO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21;SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO:26; SEQ ID NO: 27; and SEQ ID NO: 28.

C. NAABs for PITC-Derivatized Lysine

The phenyl isothiocyanate (PITC) reagent used to activate peptideN-termini for stepwise degradation also reacts with the Nε atom in thelysine side chain. As a result, domains derived from lysine RNAsynthetase (LysRS) proteins cannot be used for specific recognition ofmodified lysine. A NAAB that is specific for PITC-derivatized lysine istherefore required. The class II RS for pyrrolysine (PylRS) served as astarting point for development. Pyrrolysine is a lysine derivative thatpossesses a pyrrole ring attached to the Nε atom by an amide linkage(Structure A). Crystal structures have been determined for PylRS boundto several ligands (23), one of which is one bond longer thanpyrrolysine (Structure B), and possesses steric similarity to a model ofPITC-derivatized lysine (Structure C).

Genomic DNA for the archaea Methanosarcina mazei, the source organismfor the crystal structure, will be obtained from the American TypeCulture Collection (ATCC). The gene will be cloned and expressed. Therelevant substrate for assessing compatibility with the DAPES strategyis a peptide with an N-terminal lysine that has been modified with PITCon its side chain, but not its amino terminus. It is expected that theside chain will be derivatized during previous cycles, but that theN-terminus will be regenerated by the cleavage step of the precedingcycle. A peptide with the sequence DKGMMGSSC will be obtained. Thepeptide will be derivatized with PITC, modifying both the N-terminus andthe side chain of the lysine at the second position. The modifiedaspartate residue will be with the designed enzyme, which has excellentactivity against PITC-modified aspartate. The resulting peptide, with anN-terminal lysine modified only on its side chain, will be purified fromthe reaction mixture by HPLC. The peptide will then be immobilized onthe nanogel surface via its C-terminal cysteine. The liberated PylRSdomain will be fluorescently labeled with Cy5 and assayed for binding tothe immobilized peptide.

In the event that the engineered domain exhibits poor binding, astructural model of the NAAB in complex with pyrrolysine will beconstructed using the crystal structure as a template. Computationaldesign will be performed with the program RosettaDesign (24) to optimizethe shape complementarity between the protein and the amino acid. Wewill introduce the suggested mutations into the gene for the NAAB,express and purify the protein, and reassess the binding properties ofthe new mutant NAAB.

D. NAABs for Phosphorylated Amino Acids

In accordance with various aspects of the present invention, the NAABsmay also include reagents capable of specifically binding tophosphorylated N-terminal amino acids (e.g., phosphotyrosine,phosphoserine, and phosphothreonine).

The proteome is elaborated by post-translational modifications. Thesemarks are reversible and provide a snapshot of the current state of acell with respect to signaling pathways and other regulatory control.Side chain phosphorylation, which primarily occurs on tyrosine, serine,and threonine residues, is a well-known post-translational modification.However, characterization of phosphorylated amino acids by massspectrometry is difficult. Phosphate groups can be altered or lostduring the ionization process, and sample enrichment is typicallyrequired to cope with issues of dynamic range (2). Identification ofphosphorylated amino acids using digital protocols (e.g., DAPES) isimproved because of the improved dynamic range and mild bufferconditions afforded by the present invention. Moreover, the ability todistinguish between phosphorylated and unphosphorylated amino acidscould have a huge impact for characterizing cellular and disease states.

NAABs that specifically bind to either phosphoserine, phosphotyrosine,or phosphothreonine can be made by modifying certain tRNA synthetases toinclude one or more mutations. For example, methanogenic archaea possessan RS for phosphoserine. In contrast to most organisms, methanogenicarchaea lack a CysRS. In these organisms, phosphoserine (Sep) is firstligated to the tRNA for cysteine, and then converted to Cys-tRNA in asubsequent step. A crystal structure of SepRS, a class II synthetase incomplex with Sep is available from the PDB (pdb code: 2DU3 (36)).

While there are no known phosphotyrosine tRNA synthetases, RSs forseveral chemically similar analogs have been obtained via directedevolution (37-39). The class I TyrRS from Methanococcus jannaschii isthe parental protein for these mutants, and a crystal structure isavailable for engineering (pdb code: 1U7D (apo), 1J1U(holo)). There areseveral relevant mutant RSs, most notably for sulfotyrosine (37),p-acetyl-L-phenylalanine (pAF), and p-carboxymethyl-L-phenylalanine(pCMF).

Given the stereochemical similarity between phosphate and sulfate, andthe fact that phosphatases and phosphoryltransferases often acceptsulfates and sulfuryl groups as substrates (40), it has been found thatthe sulfotyrosine RS will recognize phosphotyrosine without furthermodification. The pAF RS, for which a crystal structure is available(pdb code: 1ZH6), differs from the sulfotyrosine RS at only two residues(38). Thus, if necessary a template is available for structural modelingand further protein engineering.

There are no reported pThrRSs or previously engineered RSs thatrecognize pThr analogs. Consequently, generation of a pThrRS may requiremore extensive protein engineering. We will approach this task from twodirections. First, we will use computational design to widen the bindingpocket of SepRS to accommodate the additional methyl group present inpThr. Second, we will use the motif-directed design approach to graftpreviously observed phosphate-binding interactions into the bindingpocket of ThrRS. The PDB contains hundreds of examples of bindinginteractions involving phosphotyrosine (308 examples), phosphoserine(385), and phosphothreonine (325) that are suitable for building a motiflibrary of protein-phosphate interactions. The same design protocolsuccessfully used to switch the specificity of eMAP to eLAP will beapplied to transplant these interaction motifs into E. coli ThrRS.Mutagenesis of SepRS and ThrRS proteins will be performed using theQuikChange protocol. We will purchase a peptide with the sequencepTGMMGSSC for attachment to the nanogel surface and characterization ofbinding by single-molecule detection.

It is expected that a NAAB for pThr may also bind to N-terminal pSer. Ifso, this NAAB can be used for pThr and pSer, and then the specific aminoacid can be inferred by evaluating the surrounding sequence to map thepeptide onto a reference proteome library. Alternatively, if de novo,phosphorylation-sensitive sequencing is required, then the efficacy ofapplying a pSer NAAB, detecting binding, then applying a pThr NAABwithout an intervening wash step will be assessed. Bound pSer terminiwill be blocked by the pSer NAAB, and only additional fluorescent spotswill be identified as pThr residues.

E. Fluorophores

In accordance with various aspects of the present invention, the NAABsare fluorescently labeled such that when a NAAB binds to an amino acid,fluorescence can be detected. Fluorophores useful for fluorescentlylabels on the NAABs include, for example, but are not limited to Cy3 andCy5. The fluorophores are usually coupled on-specifically to free aminegroups (e.g., lysine side chains) of the NAABs.

II. Method of Making NAABs by Introducing Mutations into tRNA SynthetaseProteins

The present invention also relates to a method for making a NAAB byintroducing mutations into the amino acid sequence of a tRNA synthetase(RS) to produce a NAAB that selectively binds to a particular N-terminalamino acid. For example, such methods can involve introducing one ormore mutations into a naturally occurring RS (e.g., into a wild-type E.coli RS). Such methods can also involve introducing one or moreadditional mutations into an RS that already includes one or more aminoacid mutations in its sequence as compared to the sequence of acorresponding wild-type RS.

The methods for making NAABs comprise identifying the amino acid bindingdomain of a tRNA synthetase, introducing one or more mutations into theamino acid binding domain to create a NAAB, and assaying the NAAB forspecific binding to an N-terminal amino acid of a polypeptide.

Where the tRNA synthetase is a class I tRNA synthetase, identificationof the amino acid binding domain can be accomplished, for example, byconstructing a sequence alignment that aligns pairwise the amino acidsequences of two or more class I tRNA synthetases with one another,wherein one of the class I tRNA synthetases has a previously definedamino acid binding domain. This allows for identification ofcorresponding sequence positions between proteins in order to shareuseful mutations between NAABs. Thus, in certain aspects of thesemethods, the tRNA synthetase is a first class I tRNA synthetase and theidentifying step comprises aligning an amino acid sequence of the firstclass I tRNA synthetase with an amino acid sequence of a second class ItRNA synthetase having a previously defined amino acid binding domain.For example, the amino acid binding domain of E. coli MetRS is known tobe encompassed within amino acids 4 to 547 of the protein. Thus, theamino acid sequence of the second class I tRNA synthetase can comprisethe amino acid sequence of full-length E. coli MetRS (SEQ ID NO: 5) or afragment thereof which includes the amino acid binding domain. Inaddition, the amino acid sequence of the second class I tRNA synthetasecan comprise a wild-type sequence or can comprise a sequence containingone or more mutations, so long as the presence of the mutations does notsignificantly impair the ability of the sequence to align with otherclass I tRNA synthetases. For example, the amino acid sequence of thesecond class I tRNA synthetase can comprise the amino acid sequence ofthe engineered MetRS fragment described above (of SEQ ID NO: 4), whichcontains four amino acid substitutions as compared to the correspondingfragment of wild-type E. coli MetRS. The identifying step can comprisealigning the amino acid sequence of full-length E. coli MetRS (SEQ IDNO: 5) or a fragment thereof which includes the amino acid bindingdomain with a class I tRNA synthetase selected from the group consistingof arginine, cysteine, glutamate, glutamine, isoleucine, leucine,lysine, methionine, tyrosine, tryptophan, and valine.

The method can also involve constructing a multiple sequence alignmentthat aligns the amino acid sequences of the first class I tRNAsynthetase, the second class I tRNA synthetase, and at least oneadditional class I tRNA synthetase. For example, the multiple sequencealignment can align the sequences of at least five, at least seven, orat least nine class I tRNA synthetases. Thus, the multiple sequencealignment can align the amino acid sequence of full-length E. coli MetRS(SEQ ID NO: 5) or a fragment thereof which includes the amino acidbinding domain with the amino acid sequences of at least two other classI tRNA synthetases selected from the group consisting of arginine,cysteine, glutamate, glutamine, isoleucine, leucine, lysine, methionine,tyrosine, tryptophan, and valine.

Following alignment of an amino acid sequence of a first class I tRNAsynthetase with an amino acid sequence of a second class I tRNAsynthetase having a previously defined amino acid binding domain, theboundaries of the amino acid binding domain of the first class I tRNAsynthetase can be identified using the known boundaries of the aminoacid binding domain in the second class I tRNA synthetase as a guide.

Once the amino acid binding domain of a given class I tRNA synthetasehas been identified, mutations homologous to the four substitutionmutations present in the engineered MetRS fragment described above areintroduced into the amino acid binding domain of the class I tRNAsynthetase. Thus, for each class I tRNA synthetase, the leucine atposition 13 of wild-type E. coli MetRS is replaced with a serine (L13S),the phenylalanine at position 260 is replaced with a leucine (Y260L),the aspartic acid at position 296 is replaced with a glycine (D296G),and the histidine at position 301 is replaced with a leucine (H301L).

The binding affinity of each NAAB containing these mutations against apanel of N-terminal amino acids can be predicted in silica using acomputer modeling program (e.g., the Rosetta modeling program). Any NAABwith significant predicted cross-binding with undesired target peptidescan be subjected to computational redesign for specificity using amulti-state strategy (11). For example, the computational redesign mayidentify one or more additional mutations likely to improve the bindingspecificity of the NAAB for a particular N-terminal amino acid. In thisapproach, structural models of the NAAB in complex with both the desiredand undesired amino acids are constructed in silico.

If computational redesign identifies any further mutations as beinglikely to improve the binding specificity of the NAAB for a particularN-terminal amino acid, such mutations can be introduced into the NAAB.

Similar methods can be used to identify the amino acid binding domainsof the class II RSs and introduce mutations into those domains toproduce NAABs that selectively bind to N-terminal amino acids that areactivated by class II RSs (Ala, Pro, Ser, Thr, His, Asp, Asn, Lys, Gly,and Phe).

The catalytic domain of class II RS proteins contains the amino acidspecificity for the enzyme, and these domains can be used as a startingpoint for developing additional NAABs. Although class II RSs function asmultimers, the catalytic domain of the HisRS from E. coli can be mademonomeric by liberating it from its activation domain (20). The crystalstructure of the enzyme in complex with histidyl-adenylate is available(pdb code 1KMM (21)), and can serve as a basis for computationalstructure-based design. At least one RS crystal structure is availablefor each of the amino acids activated by class II RSs (Ala, Pro, Ser,Thr, His, Asp, Asn, Lys, Gly, and Phe).

For example, the amino acid binding domains for each of the class II RSscan be identified using the monomeric fragment of E. coli HisRS (SEQ IDNO: 9) as a guide to identify corresponding domains in other class IIRSs. Structural alignments between the monomeric fragment of E. coliHisRS (residues 3-180 and corresponding domains in other class II RSscan be obtained from the Dali web server (22). Multiple sequencealignments for the conserved class II catalytic domain can be obtainedfrom the Pfam database (19). Using these alignments, boundaries for theamino acid binding domains for class II RSs can be identified.

Thus, in some aspects of the method of a making a NAAB, the tRNAsynthetase is a first class II tRNA synthetase and the step ofidentifying the amino acid binding domain comprises aligning an aminoacid sequence of the first class II tRNA synthetase with an amino acidsequence of a second class II tRNA synthetase having a previouslydefined amino acid binding domain. The amino acid sequence of the secondclass II tRNA synthetase can comprise the amino acid sequence amonomeric fragment of E. coli HisRS that contains the amino acid bindingdomain (e.g., SEQ ID NO: 9). The amino acid sequence of the second classII tRNA synthetase can comprise a wild-type sequence or can comprise asequence containing one or more mutations, so long as the presence ofthe mutations does not significantly impair the ability of the sequenceto align with other class I tRNA synthetases.

For example, the identifying step can comprise aligning the amino acidsequence of the monomeric fragment of E. coli HisRS with a correspondingdomain of a class II tRNA synthetase selected from the group consistingof AlaRS, ProRS, SerRS, ThrRS, AspRS, AsnRS, LysRS, GlyRS, and PheRS.

The identifying step can also comprise constructing a multiple sequencealignment that aligns the amino acid sequences of the first class IItRNA synthetase, the second class II tRNA synthetase, and at least oneadditional class II tRNA synthetase. For example, the multiple sequencealignment can align the sequences of at least five, at least seven, orat least nine class II tRNA synthetases. The multiple sequence alignmentcan align the amino acid sequence of a monomeric fragment of E. coliHisRS that contains the amino acid binding domain with a correspondingdomain of at least two other class II tRNA synthetases selected from thegroup consisting of AlaRS, ProRS, SerRS, ThrRS, AspRS, AsnRS, LysRS,GlyRS, and PheRS. Alternatively, the multiple sequence alignment canalign the amino acid sequence of a monomeric fragment of E. coli HisRSthat contains the amino acid binding domain with corresponding domainsof AlaRS, ProRS, SerRS, ThrRS, AspRS, AsnRS, LysRS, GlyRS, and PheRS.

Once the amino acid binding domain of a given class II tRNA synthetasehas been identified, mutations (e.g., substitution mutations) areintroduced into the amino acid binding domain in order to increase thebinding affinity of the domain for a particular N-terminal amino acid.

As with the methods involving class I tRNA synthetases, the methodsinvolving class II tRNA synthetases can also further comprise using acomputer modeling program to predict the binding affinity of the NAABagainst a panel of N-terminal amino acids. In addition, the NAAB can besubjected to computational redesign to identify one or more additionalmutations to improve the binding specificity of the NAAB for aparticular N-terminal amino acid. Any additional mutations identifiedusing computational redesign can then be introduced into the NAAB.

The NAABs designed and made using any of the above methods can clonedinto an expression vector, expressed in a host cell (e.g., in an E. colihost cell), purified, and assayed for specific binding to an N-terminalamino acid of a polypeptide. For example, the binding activity for eachNAAB can be assayed against a standard set of polypeptides havingdifferent N-terminal residues (e.g., custom synthesized peptides of theform XGMMGSSC, where X is a variable position occupied by each of thetwenty amino acids).

For NAABs derived from class II tRNA synthetases, if any of the E. coliprotein fragments prove to are insoluble or perform poorly as NAABs,protein design can be used to redesign hydrophobic residues that becomeexposed upon monomerization. If a crystal structure is unavailable forthe E. coli protein, a synthetic gene for an RS with an experimentallydetermined structure can be obtained. The availability of structures forthese proteins allows application of protein surface redesign if thedomain truncation results in loss of solubility, binding pocket redesignfor enhanced affinity if binding is weak, or multi-state design forenhanced specificity if promiscuous binding is observed (11).

In any of the above methods, the tRNA synthetase amino acid sequencescan be E. coli tRNA synthetase amino acid sequences.

In addition, in any of the above methods, the sequences can be alignedpairwise by various methods known in the art, for example, using thehidden Markov models available in the Pfam database (19), dynamicprogramming, and heuristic methods like BLAST.

Also, in any of the above methods, mutations that favor desired bindingand disfavor undesired binding can be introduced into any of thewild-type proteins described above by various methods, for example,using mutagenic primers to introduce mutations via site-directedmutagenesis, PCR-based mutagenesis and Kunkel mutagenesis. Variouscomputer programs can be used to design suitable primers (e.g., theQUICKCHANGE (Aligent Technologies) primer design program).

III. Polypeptide Sequencing Using NAABs

In accordance with various aspects of the present invention, the NAABsdiscussed above are used as reagents in a method of polypeptidesequencing. Generally, the method of sequencing a polypeptide comprisesthe steps of:

-   -   (a) contacting the polypeptide with one or more fluorescently        labeled N-terminal amino acid binding proteins (NAABs);    -   (b) detecting fluorescence of a NAAB bound to an N-terminal        amino acid of the polypeptide;    -   (c) identifying the N-terminal amino acid of the polypeptide        based on the fluorescence detected;    -   (d) removing the NAAB from the polypeptide;    -   (e) optionally repeating steps (a) through (d);    -   (f) cleaving the N-terminal amino acid of the polypeptide via        Edman degradation; and    -   (g) repeating steps (a) through (0 one or more times.

In step (a), the polypeptide is contacted with one or more NAABs. Invarious aspects, the polypeptide is contacted with a single type of NAABthat selectively binds to a single type of N-terminal amino acid residue(e.g., a NAAB that selectively binds to N-terminal alanine residues or aNAAB that selectively binds to N-terminal methionine residues). In otherembodiments, the polypeptide is contacted with a mixture of two or moretypes of NAABs that each selectively binds to different amino acidresidues. For example, the mixture may comprise two NAABs such as a NAABthat selectively binds to N-terminal alanine residues and a NAAB thatselectively binds to N-terminal cysteine residues. A mixture comprisingtwo or more NAABs that selectively bind to different amino acid residuesis especially useful when sequencing several polypeptidessimultaneously. Introducing multiple different NAABs also reducessequencing time because multiple N-terminal amino acid residues can beidentified during a single iteration of steps (a) through (d). As such,in various embodiments, the method comprises sequencing a plurality ofpolypeptides. These embodiments are especially suited for highthroughput sequencing methods.

In various aspects of the invention, the polypeptide may be immobilizedon a substrate prior to contact with the one or more NAABs. The peptidemay be immobilized on any suitable substrate. For example, nanogelsubstrates have been developed with low non-specific adsorption ofproteins and the ability to visualize single attached molecules on thissurface (8, 9). Moreover, a plurality of polypeptides may be immobilizedon the substrate for sequencing. Immobilizing a plurality ofpolypeptides is especially suited for high throughput sequencingmethods.

The NAABs of the present inventions are fluorescently labeled with afluorophore such that when a NAAB binds to a N-terminal amino acid,fluorescence emitted by the fluorophore can be detected by anappropriate detector. Suitable fluorophores include, but are not limitedto Cy3 and Cy5. Fluorescence can suitably be detected by detectors knownin the art. Based on the fluorescence detected, the N-terminal aminoacid of the polypeptide can identified.

In aspects of the method where the contacting step comprises contactingthe polypeptide with a mixture of two or more types of NAABs that eachselectively binds to different amino acid residues, each type of NAAB issuitably labeled with different fluorophores having differentfluorescence emission spectra. For example, the contacting step cancomprise contacting the polypeptide with a first type of NAAB and asecond type of NAAB, wherein the first type of NAAB selectively binds toa first type of N-terminal amino acid residue and the second type ofNAAB selectively binds to a second type of N-terminal amino acid residuedifferent from the first type of N-terminal amino acid residue. In suchmethods, the first type of NAAB is suitably coupled to a firstfluorophore and the second type of NAAB is suitably coupled to a secondfluorophore, wherein the first and second fluorophores have differentfluorescence emission spectra.

In step (d), the one or more NAABs are removed from the polypeptide(s).Removing the one or more NAABs includes removing any excess NAABspresent in solution and/or removing any NAABs that are bound toN-terminal amino acids of the polypeptides. Removal of the NAABs issuitably accomplished by washing the polypeptide with a suitable washbuffer in order to cause dissociation of any bound NAABs. In embodimentswhere the polypeptide is immobilized on a solid substrate, the reagentmay be removed by contacting the substrate with a suitable wash buffer.

Steps (a)-(d) may be repeated any number of times until the N-terminalamino acid of the polypeptide has been identified. In embodiments wherea plurality of polypeptides is being sequenced, steps (a)-(d) may berepeated any number of times until all of the N-terminal amino acids ofthe polypeptide(s) have been identified. During each repetition, adifferent NAAB or a set of NAABs may be used in step (a) to probe theN-terminal amino acid of the polypeptide(s). Thus, for example, wherestep (a) comprises contacting the polypeptide with a single type of NAABthat selectively binds to a single type of N-terminal amino acidresidue, it may be necessary to repeat steps (a) through (d) up to 24 ormore times in order to probe the polypeptide with a NAAB specific foreach of the twenty standard amino acids, for PITC-derivatized lysine,and for each of the three common phosphorylated amino acids.Alternatively, where step (a) comprises contacting the polypeptide withtwo or more different types of NAABs simultaneously, fewer repetitionsof steps (a) through (d) will be necessary to identify the N-terminalamino acid of the polypeptide.

After the N-terminal amino acid has been identified or after all of theN-terminal amino acids have been identified (when sequencing multiplepolypeptides simultaneously), the N-terminal amino acid(s) may becleaved from the polypepitde(s) via Edman degradation. Generally, theEdman degradation comprises reacting the N-terminal amino acid of thepolypeptide with phenyl isothiocyanate (PITC) to form a PITC-derivatizedN-terminal amino acid, and cleaving the PITC-derivatized N-terminalamino acid. In various aspects of the invention, the modified N-terminalamino acid may be cleaved using an Edman degradation enzyme as describedin further detail below. In other embodiments, the modified N-terminalamino group may be cleaved by methods known in the art including contactwith acid or exposure to high temperature. In these aspects, anysubstrate comprising the immobilized polypeptide(s) should be compatiblewith the acidic conditions or high temperatures.

FIG. 1 provides a diagrammatic representation of the steps of a methodof polypeptide sequencing according to the present invention. In step 1of FIG. 1, multiple polypeptide molecules are immobilized on asubstrate. The individual peptide molecules are suitably spatiallysegregated on the substrate. Analyte proteins may be fragmented into twoor more polypeptides prior to immobilization on the substrate.

In step 2 of FIG. 1, the immobilized polypeptides are contacted with afluorescently labeled NAAB and fluorescence of the NAAB bound to theN-terminal amino acid of any of the peptides is detected. An image ofthe substrate is suitably captured at this stage. Subsequently, the NAABis washed off the substrate. This cycle of binding, detection, andremoval of the NAAB is repeated until the N-terminal amino acids of allof the immobilized polypeptides have been identified (step 3). Next, instep 4, the N-termini of the polypeptides are reacted with phenylisothiocyanate (PITC) (black ovals in FIG. 1). In step 5, an Edmandegradation (“Edmanase”), catalyzes the removal of the PITC-derivatizedN-terminal amino acid under mild conditions. In each complete cycle, oneamino acid is sequenced from each peptide and a new N-terminus isgenerated for identification in subsequent cycles (step 6).

In the polypeptide sequencing methods described herein, some of theNAABs may bind smaller, sterically similar off-target amino acids. Forexample, the isoluecine-specific NAAB derived from IleRS and thethreonine-specific NAAB derived from ThrRS may bind N-terminal valineand serine residues, respectively, in addition to their desired targets.However, this does not hinder the effectiveness of this proteinsequencing technique. Although various aspects of the present inventionrelate to a reagent comprising NAABs for all twenty amino acids, theoptimal set size for actual sequencing may be less than twenty. Reducingthe number of NAABs involves trading off absolute specificity for fewerbinding molecules by using a reduced alphabet for protein sequences. Itmay be more efficient to identify multiple amino acids (such asisoleucine and valine) with a single NAAB, and treat these amino acidsas interchangeable when matching against a sequence database. It is alsopossible to enforce specificity in digital protocols such as DAPES byintroducing the NAABs in a step-wise fashion. For example, thevaline-specific NAAB derived from ValRS can be added before theisoleucine-specific NAAB derived from IleRS, with the intention ofidentifying and capping N-terminal valine residues before moleculesintended to target isoleucine residues that can bind to them.

Methods of the present invention possess attractive features relative tomass spectrometry. Because detection operates at the single moleculelevel, this method will have excellent dynamic range, and will beappropriate for extremely small amounts of sample. Furthermore, thedigital nature of the detection produces inherently quantitative data.Finally, because all steps can be carried out in neutral aqueous buffer,post-translation modifications (e.g., phosphorylations) remain stableand available for analysis.

IV. Enzymatic Edman Degradation

In another aspect, the present invention is directed to a method andreagents for enzymatic Edman degradation (i.e., cleaving the N-terminalamino acid of a polypeptide). In accordance with this aspect, one ormore enzymes are provided that catalyze the cleavage step of the Edmandegradation in aqueous buffer and at neutral pH, thereby providing analternative to the harsh chemical conditions typically employed inconventional Edman degradation. In one aspect, the Edman degradationenzyme a modified cruzain enzyme. Cruzian is a cysteine protease in theprotozoa Trypanosoma cruzi and was discovered to possess many of thedesired characteristics for creating an Edman degradation enzyme.

In conventional Edman degradation, polypeptides are sequenced bydegradation from their N-terminus using the Edman reagent, phenylisothiocyanate (PITC). The process requires two steps: coupling andcleavage. In the first step (coupling), the N-terminal amino group of apeptide reacts with phenyl isothiocyanate to form a thiourea. In thesecond step, treatment of the thiourea with anhydrous acid (e.g.,trifluoroacetic acid) results in cleavage of the peptide bond betweenthe first and second amino acids. The N-terminal amino acid is releasedas a thiazolinone derivative. The thiazoline derivative may be extractedinto an organic solvent, dried, and converted to the more stablephenylthiohydantoin (PTH) derivative for analysis. The most convenientmethod for identifying the PTH-amino acids generated during eachsequencing cycle is by UV absorbance and HPLC chromatography. Each aminoacid is detected by it UV absorbance at 269 nm and is identified by itscharacteristic retention time.

In digital protocols, such as DAPES, the N-terminal amino acid hasalready been identified. Therefore, there is no need to generate ordetect a phenylthiohydantoin derivative of the terminal amino acid.However, the strongly acidic conditions typically used in the cleavagestep of conventional Edman degradation protocols are incompatible withthe substrate surface upon which the polypeptides are immobilized forsingle molecule protein detection (SMD) (e.g., a nanogel surface). Onemodification of the conventional Edman degradation dispenses with theacidic conditions promotes cleavage with elevated temperature (e.g.,70-75° C.) instead (25). However, some substrate surfaces used toimmobilize peptides include bovine serum albumin (BSA), which has amelting temperature of approximately 60° C. in the absence ofstabilizing additives (26). Further, repeated cycles of heating andcooling of the substrate surface (e.g., nanogel) may be undesirable.Thus, the present invention provides a method of performing the Edmandegradation which dispenses with both acidic conditions and elevatedtemperature. Advantageously, an enzyme has been developed whichaccomplishes the cleavage step in a neutral, aqueous buffer. This enzymeavoids acidic conditions and high temperatures and decreases the cycletime for polypeptide sequencing by reducing or eliminating the need tochange buffer and temperature conditions repeatedly.

The Edman degradation enzyme (or “Edmanase”) according to the presentinvention accomplishes the chemical step of the N-terminal degradationby nucleophilic attack of the thiourea sulfur atom on the carbonyl groupof the scissile peptide bond. As noted, the enzyme was made by modifyingcruzain, a cysteine protease from the protozoa Trypanosoma cruzi (SEQ IDNO: 30). Cruzain prefers hydrophobic amino acids at the S2 positionrelative to the scissile bond, which corresponds to the phenyl moiety ofthe Edman reagent. The protease is relatively insensitive to theidentity of the amino acid at the S1 position (29), allowing forpromiscuous cleavage of diverse N-terminal residues. Furthermore, thisprotein has been the subject of extensive structural characterization(27).

The Edman degradation enzyme differs from the wild-type of cysteineprotease cruzain at four positions. One mutation (C25G) removes thecatalytic cysteine residue while three mutations (G65S, A138C, L160Y)were selected to create steric fit with the phenyl moiety of the Edmanreagent (PITC). FIG. 4A-4B depicts latter three mutations (indicated bythe arrows) introduced into a model of cruzain (pdb code: 1U9Q (27); SEQID NO: 30) to accommodate the phenyl moiety of the Edman reagent phenylisothiocyanate. FIG. 4A depicts a model for the cleavage intermediate ofan N-terminal alanine residue in the active site cleft. In addition tothe engineered residues, two wild-type residues (shown in green sticks)contribute to forming a complementary pocket. FIG. 4B depicts aspace-filling representation of the packing of the phenyl ring byprotein side chains. The methyl group of the ligand (in gray at the topof the panel) corresponds to the side chain of the N-terminal residue tobe cleaved, and is not involved in the tight packing between enzyme andsubstrate. The enzyme was expressed and purified.

Accordingly, one aspect of the present invention relates to an isolated,synthetic, or recombinant Edman degradation enzyme comprising an aminoacid sequence having a glycine residue at a position corresponding toposition 25 of wild-type Trypanosoma cruzi cruzian; a serine residue ata position corresponding to position 65; a cysteine residue at aposition corresponding to position 138; and a tryptophan residue at aposition corresponding to position 160.

The remaining amino acid sequence of the Edman degradation enzymecomprises a sequence similar to that of wild-type Trypanosoma cruzicruzian, but may contain additional amino acid mutations (includingdeletions, insertions, and/or substitutions, so long as such mutationsdo not significantly impair the ability of the Edman degradation enzymeto cleave PITC-derivatized N-terminal amino acids. For example, theremaining amino acid sequence can have at least about 80%, or at least85%, at least 90%, at least 93%, at least 95%, at least 96%, at least87%, at least 98%, or at least 99% sequence identity with the sequenceof the wild-type Trypanosoma cruzi cruzian.

In some aspects of the invention, the Edman degradation enzyme comprisesthe sequence of SEQ ID NO: 29. For example, the Edman degradation enzymecan consist of the sequence of SEQ ID NO: 29.

In various aspects of the invention, the reagents for enzymatic Edmandegradation comprise two or more enzymes. For example, one point ofconcern is the ability to cleave proline residues. If a single mutant ofcruzain cannot accomplish this reaction, then an additional enzyme wouldbe required. Naturally occurring enzymes cleave dipeptides of the formXaa-Pro from the N-terminus of peptides, for example, quiescent cellproline dipeptidase (QPP) (35), and Xaa-Pro amino peptidase (pdb code:30VK). PITC-coupled N-terminal proline is chemically and sterically verysimilar to a dipeptide. Therefore, these enzymes are excellent startingpoints for engineering a proline-specific activity.

When introducing elements of the present invention or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

As various changes could be made in the above products, compositions andprocesses without departing from the scope of the invention, it isintended that all matter contained in the above description and shown inthe accompanying drawings shall be interpreted as illustrative and notin a limiting sense.

Examples

The following non-limiting examples are provided to further illustratethe present invention.

Example 1 eLAP: A NAAB that Specifically Binds to N-Terminal LeucineResidues

In this example, an E. coli methionine aminopeptidase (eMAP) wasmodified to create a NAAB that binds specifically to N-terminal leucineresidues. Two mutually compatible leucine-contacting interactions wereidentified from the protein data bank (PDB) (15) that could beincorporated into the eMAP structure. The surrounding protein residuesof eMAP were redesigned around these two interactions. The resultingNAAB for leucine (eLAP) has 19 amino acid mutations relative to eMAP.

The eMAP and eLAP proteins were expressed and assayed for bindingagainst a panel of peptides with different N-termini. The NAAB forN-terminal leucine amino acids was non-specifically labeled with Cy5fluorophore on lysine side chains. Synthetic peptides with eitherN-terminal methionine, leucine, or asparagine amino acids were coupledto a nanogel surface by thiol linkage. An additional experiment wasperformed with no peptide added. The labeled NAAB was briefly incubatedwith the immobilized peptide, and unbound protein was removed bywashing. Bound protein, which may be bound specifically to peptides ornon-specifically to the surface, was imaged by total internal reflectionfluorescence (TIRF) microscopy. Spots exceeding a detection thresholdwere deemed to indicate bound protein and were converted to a number ofcounts per field-of-view. FIGS. 2A-2B show the binding specificity ofwild-type E. coli methionine aminopeptidase (eMAP) and eLAP in asingle-molecule detection experiment. In FIG. 2A, fluorescently labeledeMAP and eLAP NAABs were visualized after binding to immobilizedpeptides with different N-terminal amino acids. FIG. 2B depictshistograms of quantitative binding. Digital analysis of NAAB binding foreMAP and eLAP showed that each NAAB was specific for the expectedN-terminal amino acid. Both proteins exhibited roughly a 2:1 ratio ofspecific to non-specific binding.

These results demonstrate that individual N-terminal amino acids can beidentified in an SMD format using NAABs that are selective for aparticular amino acid.

Example 2 A NAAB that Specifically Binds to N-Terminal MethionineResidues

In this example, a truncated version of wild-type E. coli methionyl-tRNAsynthetase (MetRS) from E. coli, was modified to make a NAAB that bindsspecifically to N-terminal methionine residues. A truncated version ofMetRS (residues 1-547) having three amino acid mutations (L13S, Y260L,and H301L) that had been shown to pre-organize the binding site towardsthe methionine-bound conformation was obtained (16). A crystal structureis available of this mutant bound to free methionine (pdb code: 3h99).An additional mutation (D296G) was introduced to provide a more openbinding pocket capable of accommodating a peptide and avoid stericclashes. This mutation was introduced into MetRS and the altered proteinwas expressed in E. coli. The gene encoding MetRS from genomic DNA wasamplified and was cloned into the pET42a expression vector between theMfeI and XhoI sites. This yielded a genetic fusion of athrombin-cleavable GST tag and MetRS. The mutations were introducedusing the QuikChange protocol. The proteins were expressed at 16° C.overnight using the autoinduction protocol of Studier (17). TheGST-MetRS fusion was purified from lysates by affinity chromatographyusing GSTrap columns on a Bio-Rad liquid chromatography system.Following purification, proteins were labeled with Cy5 fluorophore onlysine side chains for single-molecule binding assays.

Using an SMD assay we then tested the specificity of our mutant MetRSfor peptides with different amino acids at the N-terminus. Peptides ofthe form XGMMGSSC were purchased commercially, where X is methionine,leucine, or asparagine. The peptides were immobilized on a nanogelsurface via thiol linkages, and the engineered MetRS domain was appliedto the surface. Single molecule detection of bound MetRS was imaged bytotal internal reflection fluorescence (TIRF) microscopy. The resultingimages are shown in FIG. 3. Quantitation of single-molecule bindingevents yields specific to non-specific binding of ˜7:1 and ˜13:1 for thealternate amino acids. The data in FIG. 3 show that the domain exhibitsspecific binding for N-terminal methionine, indicating that engineeredRS fragments are excellent molecular reagents for DAPES and thatcomputational protein design is an efficient method for producing NAABswith specificity for particular N-terminal amino acids.

Example 3 A NAAB that Specifically Binds to N-Terminal HistidineResidues

In this example, a histidine-tRNA synthetase (HISRS) from E. coli wasmodified to create a NAAB that binds specifically to N-terminalhistidine residues. The fragment of wild-type HisRS from 1-320 was shownto be monomeric by others. After inspecting the crystal structure ofHisRS, further residues were truncated from both ends. The initialfragment tested has from Lysine3 to Alanine180. Protein design wasconducted to replace a long loop near the binding site with a shorterloop that would create a more open pocket and result in tighter bindingto N-terminal histidine residues. This involved the replacement of an 11residue loop (from Arginine113 to Lysine123) with a 4 residue turn,wherein the four residues of the inserted turn are Glycine, Asparagine,Alanine, and Proline. Thus, this NAAB comprises an internally truncatedversion of wild-type E. coli HisRS (residues 3-180; SEQ ID NO: 10)having seven fewer residues as compared to the wild-type sequence. Thesequence of this N-terminal histidine-specific NAAB is provided by SEQID NO: 9.

FIG. 7 shows that engineered HisNAAB (SEQ ID NO: 10) exhibits enhancedbinding affinity for peptides with N-terminal histidine residues ascompared to the wild-type fragment. Biolayer interferometry kineticsdata show that the engineered HisNAAB (data in open circles) bindsN-terminal histidine with the same off-rate as the wild-type fragment(SEQ ID NO: 90 (data in solid circles), but with an enhanced on-rate. Asa result, the engineered His NAAB binds with an approximately 10-foldimprovement in binding affinity.

Example 4 Purification of an Edman Degradation Enzyme

A synthetic gene containing the Edman degradation enzyme was purchasedfrom GenScript. The gene encoded a modified version of the cruzianenzyme of T. cruzi having the following substitution mutations: C25G,G65S, A138C, and L160Y.

The gene was inserted between an NdeI and an XhoI site in a pet42(a)(Novagen) expression vector and transformed into E. coli, BL-21(De3)chemically competent cells. Protein was then over-expressed followingStudier's auto-induction protocol. Bacterial cells were harvested bycentrifugation of the cell culture at 5000 rpm and 4° C. for 10 minutes.Cells were then suspended in 1×PBS with 10% glycerol and 6M guanidinechloride, pH 7.4. Cells were then lysed by sonication (15 seconds at 20%power, 8 times on ice). The cell lysate was centrifuged at 18000 rpm, 4degrees for 20 minutes. The supernatant was then filtered through a 0.2μm cellulose acetate filter. The filtered lysate was loaded onto a 5 mLHisTrap (Ni-NTA) column and washed with 5 column volumes of bindingbuffer (50 mM Tris-HCl, 150 mM NaCl, 6M guanidine chloride, 25 mMimidazole). Bound protein was then eluted in 50 mM Tris-HCl, 150 mMNaCl, 6M guanidine chloride, 500 mM imidazole. Purified fractions wereprepared for SDS-PAGE analysis by mixing 2 parts sample with 1 part 4×loading dye. Samples were analyzed on 16% SDS-PAGE precast gels, andvisualized by Coomassie staining. The purified protein was then refoldedby successive, overnight dialyses into 1×PBS containing 5M, 3M, 1M,0.5M, and 0M guanidine chloride. Protein concentration was determinedusing the calculated molar extinction coefficient and measuring the A280on an ND-8000 spectrophotometer (Thermo Fisher Scientific).

Example 5 Substrates and Inhibitors for Edman Degradation Enzyme(Edmanase)

Single amino acid, aminomethylcoumarin (AMC) containing compounds wereobtained from BAChem (Bubendorf, Switzerland). These included Arg-AMC,Asn-AMC, Phe-AMC, Met-AMC, Ala-AMC, and Pro-AMC. Phenylisothiocyanate(PITC) was purchased from Thermo-scientific and coupled to theN-terminus of each substrate by incubating for 10 minutes at roomtemperature in a 100 μL solution of acetonitrile:pyridine:water (10:5:3)with 5 μL of PITC. The derivatized substrate was then dried by rotaryevaporation and suspended in 250 μL of 1× Phosphate Buffered Saline(PBS). Inhibitor compound,1-(2-anilino-5-methyl-1,3-thiazol-4-yl)-ethanone, was ordered fromSigma-Aldrich (St. Louis, Mo.).

Example 6 Edmanase Activity Measurements

The ability of the Edman degradation enzyme to perform N-terminalcleavage on six substrates of the form Ed-X-AMC, where Ed denotes theEdman reagent, X is an amino acid from the set (Ala, Asp, Phe, Met, Pro,Arg), and AMC is the fluorogenic amidomethylcoumarin group wascharacterized. Cleavage of the X-AMC bond was monitored by theappearance of fluorescence (FIG. 6). The engineered protein displayedactivity against all six substrates to varying degrees (See Tablebelow).

All kinetic measurements were performed in a 96-well coming plate on aBioTek Synergy2 plate reader at 30 degrees. Reactions were started byadding 5-20 μL of purified enzyme to 100 μL of 10 mM substrate solution.Final enzyme concentration was between 1 nM and 100 nM, depending on theexperiment. Fluorescence of the cleaved product was measured by excitingat 370 nm (30 second intervals for 1-10 hours) and monitoring emissionsat 460 nm. A standard curve using AMC from Invitrogen was referencedquantitate the amount of product formation.

TABLE Measured kinetic rates for Edmanase Substrate (X- AMC) K_(cat)(s⁻¹) K_(m) (μM) Kcat/K_(M) Alanine 0.55 21.3 2.6 × 10⁴ Arginine 0.087167.8 5.2 × 10² Asparagine 3.6 124.5 2.9 × 10⁴ Methionine 0.54 271.8 2.0× 10³ Phenylalanine 0.47 122.8 3.8 × 10³ Proline 0.0014 252.0 5.7 × 10¹

Example 7 Inhibition of the Edman degradation enzyme by1-(2-anilino-5-methyl-1,3-thiazol-4-yl)-ethanone

Assays were conducted as described above in Example 5, with 5 μMsubstrate, 100 nM enzyme, and 500 nM-15 μM1-(2-anilino-5-methyl-1,3-thiazol-4-yl)-ethanone. Reaction velocity wasdetermined as above, plotted against the inverse of inhibitorconcentration, and fit by non-linear least squares to determine theinhibition constant.

Example 8 Cloning of Additional N-Terminal Amino Acid Binding Proteins(NAABs)

Primers specific for each NAAB were ordered from Integrated DNATechnologies. Each NAAB was then amplified from isolated, E. coligenomic DNA and transferred to a pet42a expression vector at variouspositions, depending on the gene sequence. These constructs weretransformed into either E. coli BL21(DE3) or E. coli ‘Arctic Express’competent cells for expression.

Example 9 Expression and Purification of N-Terminal Amino Acid Binders(NAABs)

Protein was over-expressed following Studier's auto-induction protocol.Bacterial cells were harvested by centrifugation of the cell culture at5000 rpm and 4° C. for 10 minutes. Cells were then suspended in 1×PBSwith 10% glycerol, pH 7.4. Cells were then lysed by sonication (15seconds at 20% power, 8 times on ice). The cell lysate was centrifugedat 18000 rpm, 4 degrees for 20 minutes. The supernatant was thenfiltered through a 0.2 um cellulose acetate filter. The filtered lysatewas loaded onto a 1 mL GSTrap column and washed with 5 column volumes ofbinding buffer (1×PBS). Bound protein was then eluted in 50 mM Tris-HCl,10 mM reduced glutathione. Purified fractions were prepared for SDS-PAGEanalysis by mixing 2 parts sample with 1 part 4× loading dye. Sampleswere analyzed on 16% SDS-PAGE precast gels, and visualized by Coomassiestaining. Protein concentration was determined using the calculatedmolar extinction coefficient and measuring the A280 on an ND-8000spectrophotometer (Thermo Fisher Scientific).

Example 10 Binding Assays

Real time binding assays between peptides and purified NAABs wereperformed using biolayer interferometry on a Blitz system (Fortebio,Menlo Park, Calif.). This system monitors interference of lightreflected from the surface of a fiber optic sensor to measure thethickness of molecules bound to the sensor surface. Sensors coated withpeptides were allowed to bind to the NAABs in 1×PBS at several differentprotein concentrations. Binding kinetics were calculated using the Blitzsoftware package, which fit the observed binding curves to a 1:1 bindingmodel to calculate the association rate constants. NAABs were allowed todissociate by incubation of the sensors in 1×PBS. Dissociation curveswere fit to a 1:1 model to calculate the dissociation rate constants.Binding affinities were calculated as the kinetic dissociation rateconstant divided by the kinetic association rate constant.

TABLE Measured Affinity Constants Glutamate 2.12 μM Phenylalanine 3.44μM Histidine 98.7 μM Methionine 1.07 μM Asparagine 754 nM Arginine 129nM Tryptophan 48.9 nM Tyrosine 57.6 μM Phosphoserine 7.72 μMPhosphotyrosine 1.07 μM Aspartate 411 nM Isoleucine 3.01 μM Leucine 1.88μM Glutamine 531 nM Serine 938 nM Threonine 1.01 μM Valine 1.22 μMLysine 2.61 μM

FIG. 8 is a full binding matrix that shows how well every engineeredNAAB protein binds to every N-terminal amino acid. Each square in thebinding matrix represents the binding affinity for a single NAAB with anN-terminal amino acid as measured by biolayer interferometry. Each rowin the matrix contains all the binding data for a single NAAB, and eachcolumn contains the binding data for a single N-terminal amino acid(shown by single-letter code). Darker squares represent tighter binding.The NAABs exhibit cross-binding for chemically similar N-terminal aminoacids. However, the set of predicted binding patterns for each aminoacid are distinct. Thus, when taken as a set, the engineered NAABproteins are capable of identifying amino acids at the N-terminus ofpeptides.

For reference, the abbreviations of the amino acids are as follows:

Three One Amino letter letter acid code code alanine ala A arginine argR asparagine asn N aspartic asp D acid asparagine asx B or aspartic acidcysteine cys C glutamic glu E acid glutamine gln Q glutamine glx Z orglutamic acid glycine gly G histidine his H isoleucine ile I leucine leuL lysine lys K methionine met M phenylalanine phe F proline pro P serineser S threonine thr T tryptophan trp W tyrosine tyr Y valine val V

TABLE A NAAB sequences SEQ ID NO: SEQ ID wild-typeMAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELD NO: 1 eMAPRICNDYIVNEQHAVSACLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIVGKPTIMGERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREYCGHGIGRGFHEEPQVLHYDSRETNVVLKPGMTFTIEPMVNAGKKEIRTMKDGWTVKTKDRSLSAQYEHTIVVTDNGCEILTLR KDDTIPAIISHDE SEQ ID eLAPMAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELE NO: 2RICWDYIVNEQHATDSLTGHNGIDGHGSISINEVVCHGVPDDAKLLKDGDIVNIDVTVRKDGFHGDTSKMFIVGKPTIMGERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREYCGHGIGRGHHEEPQVLHYDSRETNVVLKPGMTFTIEPMVNAGKKEIRTMKDGSTVKTKDRSLSAQYEHTIVVTDNGCEILTLRK DDTIPAIISHDE SEQ ID truncatedAKKILVTCALPYANGSIHLGHMLEHIQADVWVRYQRMRGH NO: 3 wild-typeEVNFICADDAHGTPIMLKAQQLGITPEQMIGEMSQEHQTDFA MetRSGFNISYDNYHSTHSEENRQLSELIYSRLKENGFIKNRTISQLY (4-547)DPEKGMFLPDRFVKGTCPKCKSPDQYGDNCEVCGATYSPTELIEPKSVVSGATPVMRDSEHFFFDLPSFSEMLQAWTRSGALQEQVANKMQEWFESGLQQWDISRDAPYFGFEIPNAPGKYFYVWLDAPIGYMGSFKNLCDKRGDSVSFDEYWKKDSTAELYHFIGKDIVYFHSLFWPAMLEGSNFRKPSNLFVHGYVTVNGAKMSKSRGTFIKASTWLNHFDADSLRYYYTAKLSSRIDDIDLNLEDFVQRVNADIVNKVVNLASRNAGFINKRFDGVLASELADPQLYKTFTDAAEVIGEAWESREFGKAVREIMALADLANRYVDEQAPWVVAKQEGRDADLQAICSMGINLFRVLMTYLKPVLPKLTERAEAFLNTELTWDGIQQPLLGHKVNPFKALYNRIDMRQVE ALVEASK SEQ ID MetAKKILVTCASPYANGSIHLGHMLEHIQADVWVRYQRMRGH NO: 4 NAAB*EVNFICADDAHGTPIMLKAQQLGITPEQMIGEMSQEHQTDFAGFNISYDNYHSTHSEENRQLSELIYSRLKENGFIKNRTISQLYDPEKGMFLPDRFVKGTCPKCKSPDQYGDNCEVCGATYSPTELIEPKSVVSGATPVMRDSEHFFFDLPSFSEMLQAWTRSGALQEQVANKMQEWFESGLQQWDISRDAPYFGFEIPNAPGKYFYVWLDAPIGLMGSFKNLCDKRGDSVSFDEYWKKDSTAELYHFIGKGIVYFLSLFWPAMLEGSNFRKPSNLFVHGYVTVNGAKMSKSRGTFIKASTWLNHFDADSLRYYYTAKLSSRIDDIDLNLEDFVQRVNADIVNKVVNLASRNAGFINKRFDGVLASELADPQLYKTFTDAAEVIGEAWESREFGKAVREIMALADLANRYVDEQAPWVVAKQEGRDADLQAICSMGINLFRVLMTYLKPVLPKLTERAEAFLNTELTWDGIQQPLLGHKVNPFKALYNRIDMRQVE ALVEASK SEQ ID wild-typeMTQVAKKILVTCALPYANGSIHLGHMLEHIQADVWVRYQR NO: 5 MetRSMRGHEVNFICADDAHGTPIMLKAQQLGITPEQMIGEMSQEH (fullQTDFAGFNISYDNYHSTHSEENRQLSELIYSRLKENGFIKNRT length)ISQLYDPEKGMFLPDRFVKGTCPKCKSPDQYGDNCEVCGATYSPTELIEPKSVVSGATPVMRDSEHFFFDLPSFSEMLQAWTRSGALQEQVANKMQEWFESGLQQWDISRDAPYFGFEIPNAPGKYFYVWLDAPIGYMGSFKNLCDKRGDSVSFDEYWKKDSTAELYHFIGKDIVYFHSLFWPAMLEGSNFRKPSNLFVHGYVTVNGAKMSKSRGTFIKASTWLNHFDADSLRYYYTAKLSSRIDDIDLNLEDFVQRVNADIVNKVVNLASRNAGFINKRFDGVLASELADPQLYKTFTDAAEVIGEAWESREFGKAVREIMALADLANRYVDEQAPWVVAKQEGRDADLQAICSMGINLFRVLMTYLKPVLPKLTERAEAFLNTELTWDGIQQPLLGHKVNPFKALYNRIDMRQVEALVEASKEEVKAAAAPVTGPLADDPIQETITFDDFAKVDLRVALIENAEFVEGSDKLLRLTLDLGGEKRNVFSGIRSAYPDPQALIGRHTIMVANLAPRKMRFGISEGMVMAAGPGGKD IFLLSPDAGAKPGHQVK SEQ IDtruncated VDVSLPGASLFSGGLHPITLMERELVEIFRALGYQAVEGPEV NO: 6 wild-typeESEFFNFDALNIPEHHPARDMWDTFWLTGEGFRLEGPLGEEV PheRSEGRLLLRTHTSPMQVRYMVAHTPPFRIVVPGRVFRFEQTDAT (86-350)HEAVFHQLEGLVVGEGIAMAHLKGAIYELAQALFGPDSKVRFQPVYFPFVEPGAQFAVWWPEGGKWLELGGAGMVHPKVFQAVDAYRERLGLPPAYRGVTGFAFGLGVERLAMLRYGIPDIR YFFGGRLKFLEQFKGVL SEQ IDPheNAAB VDVSLPGASLFSGGDHPITLMERELVEIFRALGYQAVEGPEV NO: 7 (86-350)ESEFFNFDALNIPENGPARDMWDTVGKTGEGFRLEGPDGEEVEGRLLLRTHTSPMQVRYMVAHTPPFRIVVPGRVFRAEQTDATAEAVFHQLEGLVVGEGVNEGDLYGAIYELAQALFGPDSKVRFQPVTFPFVEPGAQFAVWWPEGGKWLELGGAGMVGPNVFQAVDAYRERLGDPPAYRGVTGFAFGLGVERLAMLRYGIPD IRYF SEQ ID wild-typeMLEEALAAIQNARDLEELKALKARYLGKKGLLTQEMKGLS NO: 8 PheRSALPLEERRKRGQELNAIKAALEAALEAREKALEEAALKEAL (fullERERVDVSLPGASLFSGGLHPITLMERELVEIFRALGYQAVE length)GPEVESEFFNFDALNIPEHHPARDMWDTFWLTGEGFRLEGPLGEEVEGRLLLRTHTSPMQVRYMVAHTPPFRIVVPGRVFRFEQTDATHEAVFHQLEGLVVGEGIAMAHLKGAIYELAQALFGPDSKVRFQPVYFPFVEPGAQFAVWWPEGGKWLELGGAGMVHPKVFQAVDAYRERLGLPPAYRGVTGFAFGLGVERLAMLRYGI PDIRYFFGGRLKFLEQFKGVL SEQ IDtruncated NIQAIRGMNDYLPGETAIWQRIEGTLKNVLGSYGYSEIRLPIV NO: 9 wild-typeEQTPLFKRAIGEVTDVVEKEMYTFEDRNGDSLTLRPEGTAGC HisRSVRAGIEHGLLYNQEQRLWYIGPMFRHERPQKGRYRQFHQLG (3-180)CEVFGLQGPDIDAELIMLTARWWRALGISEHVTLELNSIGSL EARANYRDA SEQ ID HisNAABKNIQAIRGMNDYLPGETAIWQRIEGTLKNVLGSYGYSEIRLPI NO: 10 (3-180)VEQTPLFKRAIGEVTDVVEKEMYTFEDRNGDSLTLRPEGTAGCVRAGIEHGLLYNQEQRLWYIGPMFGNAPQFHQLGCEVFGLQGPDIDAELIMLTARWWRALGISEHVTLELNSIGSLEARAN YRDA SEQ ID AlaNAABSKSTAEIRQAFLDFFHSKGHQVVASSSLVPHNDPTLLFTNAG NO: 11MNQFKDVFLGLDKRNYSRATTSQRCVRAGGKHNDLENVGYTARHHTFFEMLGNFSFGDYFKHDAIQFAWELLTSEKWFALPKERLWVTVYESDDEAYEIWEKEVGIPRERIIRIGDNKGAPYASDNFWQMGDTGPCGPCTEIFYDHGDHIWGGPPGSPEEDGDRYIEIWNIVFMQFNRQADGTMEPLPKPSVDTGMGL ERIAAVLQHVNSNYDIDL SEQ ID ArgNAABEKQTIVVDYSAPNVAKEMHVGHLRSTIIGDAAVRTLEFLGH NO: 12KVIRANHVGDWGTQFGMLIAWLEKQQQENAGEMELADLEGFYRDAKKHYDEDEEFAERARNYVVKLQSGDEYFREMWRKLVDITMTQNQITYDRLNVTLTRDDVMGESLYNPMLPGIVADLKAKGLAVESEGATVVFLDEFKNKEGEPMGVIIQKKDGGYLYTTTDIACAKYRYESLHADRVLYYIDSRQHQHLMQAWAIVRKAGYVPESVPLEHHMFGMMLGKDGKPFKTRAGGTVKLADLLDETLERARRLVAEKNPDMPADELEKLANAVGIGAVKYADLSKNRTTDYIFDWDNMLAFEGNTAPYMQYAYTRVLSVFRKAEINEEQLAAAPVIIREDREAQLAARLLQFEETLTVVAREGTPHVMCAYLYDLAGLFSGFYEHCPILSAENEEVRNSRLKLAQ LTAK TLKLGLDTLGIETVERM SEQ IDAsnNAAB SIEYLREVAHLRPRTNLIGAVARVRHTLAQALHRFFNEQGFF NO: 13WVSTPLITASDTEGAGEMFRVSTLDLE NLPRNDQGKVDFDKDFFGKESFLTVSGQLNGETYACALSKIYTFGPTFRAENSNTSRHLAEFWMLEPEVAFANLNDIAGLAEAMLKYVFKAVLEERADDMKFFAERVDKDAVSRLERFIEADFAQVDYTDAVTILENCGRKFENPVYWGVDLSSEHERYLAEEHFKAPVVVKNYPKDIKAFYMRLNEDGKTVAAMDVLAPGIGEIIGGSQREERLDVLDERMLEMGLNKEDYWWYRDLRRYGTVP HSGFGLGFERLIAYVTGVQNVRDVIPFPRTPSEQ ID AspNAAB LPLDSNHVNTEEARLKYRYLDLRRPEMAQRLKTRAKITSLV NO: 14RRFMDDHGFLDIETPMLTKATPEGARDYLVPSRVHKGKFYALPQSPQLFKQLLMMSGFDRYYQIVKCFRDEDLRADRQPEFTQIDVETSFMTAPQVREVMEALVRHLWLEVKGVDLGDFPVMTFAEAERRYGSDKPDLRNPMELTDVADLLRSVEFAVFAGPANDPKGRVAALRVPGGASLTRKQIDEYDNFVKIYGAKGLAYIKVNERAKGLEGINSPVAKFLNAEHEAILDRTAAQDGDMIFFGADNKKIVADAMGALRLKVGKDLGLTDESKWAPLWVIDFPMFEDDGEGGLTAMHHPFTSPKDMTAAELKAAPENAVANAYDMVINGYEVGGGSVRIHNGDMQQTVFGILGINEEEQREKFGFLLDALKYGTPPHAGLAFGLDRLTMLLTGTDNIRDVIAFPK SEQ ID CysNAABMLKIFNTLTRQKEEFKPIHAGEVGMYVCGITVYDLCHIGHGR NO: 15TFVAFDVVARYLRFLGYKLKYVRNITDI DDKIIKRANENGESFVAMVDRMIAEMHKDFDALNILRPDMEPRATHHIAEIIELTEQLIAKGHAYVADNGDVMFDVPTDPTYGVLSRQDLDQLQAGARVDVVDDKRNPMDFVLWKMSKEGEPSWPSPWGAGRPGWHIECSAMNCKQLGNHFDIHGGGSDLMFPHHENEIAQSTCAHDGQYVNYWMHSGMVMVDREKMSKSLGNFFTVRDVLKYYDAETVRYFLMSGHYRSQLNY SEQ ID GlnNAABTNFIRQIIDEDLASGKHTTVHTRFPPEPNGYLHIGHAKSICLNF NO: 16GIAQDYKGQCNLRFDDTNPVKEDIEYVESIKNDVEWLGFHWSGNVRYSSDYFDQLHAYAIELINKGLAYVDELTPEQIREYRGTLTQPGKNSPYRDRSVEENLALFEKMRTGGFEEGKACLRAKIDMASPFIVMRDPVLYRIKFAEHHQTGNKWCIYPMYDFTHCISDALEGITHSLCTLEFQDNRRLYDWVLDNITIPVHPRQYEFSR SEQ ID GluNAABIKTRFAPSPTGYLHVGGARTALYSWLFARNHGGEFVLRIEDT NO: 17DLERSTPEAIEAIMDGMNWLSLEWDEGPYYQTKRFDRYNAVIDQMLEEGTAYKCYCSKERLEALREEQMAKGEKPRYDGRCRHSHEHHADDEPCVVRFANPQEGSVVFDDQIRGPIEFSNQELDDLIIRRTDGSPTYNFCVVVDDWDMEITHVIRGEDHINNTPRQINILKALNAPVPVYAHVSMINGDDGKKLSKRHGAVSVMQYRDDGYLPEALLNYLVRLGWSHGDQEIFTREEMIKYFTLNA VSKSASAFNTDKLLWLNHHYI SEQ IDIleNAAB FPMRGDLAKREPGMLARWTDDDLYGIIRAAKKGKKTFILHD NO: 18GPPYANGSIHIGHSVNKILKDIIIKSKGLSGYDSPYVPGWDCHGLPIELKVEQEYGKPGEKFTAAEFRAKCREYAATQVDGQRKDFIRLGVLGDWSHPYLTMDFKTEANIIRALGKIIGNGHLHKGAKPVHWCVDCRSALAEAEVEYYDKTSPSIVAFQAVDQDALKTKFGVSNVNGPISLVIWTTTPWTLPANRAISIAPDFDYALVQIDGQAVILAKDLVESMQRIGVSDYTILGTVKGAELELLRFTHPFMDFDVPAILGDHVTLDAGTGAVHTAPGHGPDDYVIGQKYGLETANPVGPDGTYLPGTYPTLDGVNVFKANDIVVALLQEKGALLHVEKMQHSYPCCWRHKTPIIFRATPQWFVSMDQKGLRAQSLKEIKGVQWIPDWGQARIESMVANRPDWCISRQRTWGVPMSLFVHKDTEELHPRTLELMEEVAKRVEVDGIQAWWDLDAKEILGDEADQYVKVPDTLDVWFDSGSTHSSVVDVRPEFAGHAADMYLEGSDQHRGWFMSSLMISTAMKGKAPYRQVLTHGFTVDGQGRKMSKSIGNTVSPQDVMNKLGADILRLWVASTDYTGEMAVSDEILKRAADSYRRIRNTARFLLANLNGFDPAKDMVKPEEMVVLDRWAVGCAKAAQEDILKAYEAYDFHEVVQRLMRFCSVEMGSFYLDIIKDRQYTAKADSVARRSCQTALYHIAEALVRWMAPILSFTADEVWGYLPGERE SEQ ID LeuNAABIESKVQLHWDEKRTFEVTEDESKEKYYCLSMLPYPSGRLHM NO: 19GHVRNYTIGDVIARYQRMLGKNVLQPIGWDAFGLPAEGAAVKNNTAPAPWTYDNIAYMKNQLKMLGFGYDWSRELATCTPEYYRWEQKCFTELYKKGLVYKKTSAVNWCPNDQTVLANEQVIDGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDTVKTMQRNWIGRSEGVEITFNVKDYDNTLTVYTTRPDTFMGCTYLAVAAGHPLAQKAAENNPELAAFIDECRNTKVAEAEMATMEKKGVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPGHDQRDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAIADKLTEMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDVVMDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESSWYYARYTCPEYKEGMLDSKAANYWLPVDIYIGGIEHAIMHLLYFRFFHKLMRDAGMVNSDEPAKQLLCQGMVLADAFYYVGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGMSKMSKSKNNGIDPQVMVERYGADTVRLFMMFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKGDVAALNVDALTEDQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIMELMNKLAKAPTDGEQDRALMQEALLAVVRMLNPFTPHICFTLWQELKG EGDIDNAPWP SEQ ID LysNAABANDKSRQTFVVRSKILAAIRQFMVARGFMEVETPMMQVIPG NO: 20GASARPFITHHNALDLDMYLRIAPELYLKRLVVGGFERVFEINRNFRNEGISVRHNPEFTMMELYMAYADYHDLIELTESLFRTLAQEVLGTTKVTYGEHVFDFGKPFEKLTMREAIKKYRPETDMADLDNFDAAKALAESIGITVEKSWGLGRIVTEIFDEVAEAHLIQPTFITEYPAEVSPLARRNDVNPEITDRFEFFIGGREIGNGFSELNDAEDQAERFQEQVNAKAAGDDEAMFYDEDYVTALEYGLPPTAGLGIGIDRMIMLFTNSHTIRDVILFPAMRP SEQ ID ProNAABMIRKLASGLYTWLPTGVRVLKKVENIVREEMNNAGAIEVLM NO: 21PVVQPSELWQESGRWEQYGPELLRIADRGDRPFVLGPTHEEVITDLIRNELSSYKQLPLNFYQIQTKFRDEVRPRFGVMRSREFLMKDAYSFHTSQESLQETYDAMYAAYSKIFSRMGLDFRAVQADTGSIGGSASHEFQVLAQSGEDDVVFSDTSDYAANIELAEAIAPKEPRAAATQEMTLVDTPNAKTIAELVEQFNLPIEKTVKTLLVKAVEGSSFPLVALLVRGDHELNEVKAEKLPQVASPLTFATEEEIRAVVKAGPGSLGPVNMPIPVVIDRTVAAMSDFAAGANIDGKHYFGINWDRDVATPEIADIRNVVAGDPSPDGQGTLLIKR GIEVGHIFQLG SEQ ID SerNAABMLDPNLLRNEPDAVAEKLARRGFKLDVDKLGALEERRKVL NO: 22QVKTENLQAERNSRSKSIGQAKARGEDIEPLRLEVNKLGEELDAAKAELDALQAEIRDIALTIPNLPADEVPVGKDENDNVEVSRWGTPREFDFEVRDHVTLGEMYSGLDFAAAVKLTGSRFVVMKGQIARMHRALSQFMLDLHTEQHGYSENYVPYLVNQDTLYGTGQLPKFAGDLFHTRPLEEEADTSNYALIPTAEVPLTNLVRGEIIDEDDLPIKMTAHTPCFRSEAGSYGRDTRGLIRMHQFDKVEMVQIVRPEDSMAALEEMTGHAEKVLQLLGLPYRKIILCTGDMGFGACKTYDLEVWIPAQNTYREISSCSNVWDFQARRMQARCRSKSDKKTRLVHTLNGSGLAVGRTLVAVMENYQQ ADGRIEVPEVLRPYMNGLEYI SEQ IDThreNAAB RDHRKIGKQLDLYHMQEEAPGMVFWHNDGWTIFRELEVFV NO: 23RSKLKEYQYQEVKGPFMMDRVLWEKTGHWDNYKDAMFTTSSENREYCIKPMNCPGHVQIFNQGLKSYRDLPLRMAEFGSCHRNEPSGSLHGLGRVRGFTQDDAHIFCTEEQIRDEVNGCIRLVYDMYSTFGFEKIVVKLSTRPEKRIGSDEMWDRAEADLAVALEENNIPFEYQLGEGAFYGPKIEFTLYDCLDRAAQCGTVQLDFSLPSRLSASYVGEDNERKVPVMIHRAILGSMEVFIGILTEEFAGFFPTWLAPVQVVIMNITDSQSEYVNELTQKLSNAGIRVKADLRNEKIGFKIREHTLRRVPYMLVCGDKEVESGKVAVRTRRG KDLGSMDVNEVIEKLQQEIRSRSLKQLEESEQ ID TrpNAAB MTKPIVFSGAQPSGELTIGNYMGALRQWINMQDDYHCIYCI NO: 24VDQHAITVRQDAQKLRKATLDTLALYLACGIDPEKSTIFVQSHVPEHAQLGWALNCYTYFGELSRMTQFKDKSARYAENINAGLFDYPVLMAADILLYQTNLVPVGEDQKQHLELSRDIAQRFNALYGDIFKVPEPFIPKSGARVMSLLEPTKKMSKSDDNRNNVIGLLEDPKSVVKKIKRAVTDSDEPPVVRYDVQNKAGVSNLL DILSAVTGQSIPELEKQ SEQ IDTyrNAAB MASSNLIKQLQERGLVAQVTDEEALVERLAQGPIALYCGFDP NO: 25TADSLHLGHLVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVDKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMINKEAVKQRLNREDQGISFTEFSYNLLQGYDFACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVKMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGRFTLLRR GKKNYCLICWK SEQ ID ValNAABMEKTYNPQDIEQPLYEHWEKQGYFKPNGDESQESFCIMIPPP NO: 26NVTGSLHMGHAFQQTIMDTMIRYQRMQGKNTLWQVGTDHAGIATQMVVERKIAAEEGKTRHDYGREAFIDKIWEWKAESGGTITRQMRRLGNSVDWERERFTMDEGLSNAVKEVFVRLYKEDLIYRGKRLVNWDPKLRTAISDLEVENRESKGSMWHIRYPLADGAKTADGKDYLVVATTRPETLLGDTGVAVNPEDPRYKDLIGKYVILPLVNRRIPIVGDEHADMEKGTGCVKITPAHDFNDYEVGKRHALPMINILTFDGDIRESAQVFDTKGNESDVYSSEIPAEFQKLERFAARKAVVAAIDALGLLEEIKPHDLTVPYGDRGGVVIEPMLTDQWYVRADVLAKPAVEAVENGDIQFVPKQYENMYFSWMRDIQDWCISRQLWWGHRIPAWYDEAGNVYVGRNEEEVRKENNLGADVALRQDEDVLDTWFSSALWTFSTLGWPENTDALRQFHPTSVMVSGFDIIFFWIARMIMMTMHFIKDENGKPQVPFHTVYMTGLIRDDEGQKMSKSKGNVIDPLDMVDGISLPELLEKRTGNMMQPQLADKIRKRTEKQFPNGIEPHGTDALRFTLAALASTGRDINWDMKRLEGYRNFCNKLWNASRFVLMNTEGQDCGFNGGEMTLSLADRWILAEFNQTIKAYREALDSFRFDIAAGILYEFTWNQFCDWYLELTKPVMNGGTEAELRGTR HTLVTVLEGLLRLAHPIIPFITETIWQSEQ ID Phospho- MDEFEMIKRNTSEIISELREVLKKDEKSALIGFEPSGKIHLGH NO: 27tyrosine YLQKKMIDLQNAGFDIIIPLADLHAYLNQKGELDEIRKIGDY NAAB**NKKVFEAMLKAKYVYGSEFQLDKYTLNVYRLALKTTLKARRSMELIAREDENPVAEVIYPIMQVNGCHYKGVDVAVGGMEQRKIMLARELLPKKVVCIHPVLTGLDGEGKMSSSGNFIAVDDSPEEIRAFKKAYCPAGVVEGNPEIAKYFLEYPLTIKPEKFGGDLTVNSYEESLFKNKELHPMDLKAVAEELIKILEPIRK SEQ ID Phospho-MRFDPEKIKKDAKENFDLTWNEGKKMVKTPTLNERYPRTTF NO: 28 serineRYGKAHPVYDTIQKLREAYLRMGFEEMMNPLIVDEKEVHK NAABQFGSEALAVLDRCFYLAGLPRPNVGISDERIAQINGILGDIGDEGIDKVRKVLHAYKKGKVEGDDLVPEISAALEVSDALVADMIEKVFPEFKELVAQASTKTLRSHMTSGWFISLGALLERKEPPFHFFSIDRCFRREQQEDASRLMTYYSASCVIMDENVTVDHGKAVAEGLLSQFGFEKFLFRPDEKRSKYYVPDTQTEVFAFHPKLVGSNSKYSDGWIEIATFGIYSPTALAEYDIPCPVMNLGLGVERLAMILHDAPDIRSLTYPQIPQYSEWEMSDSELAKQVFVDKTPETPEGREIADAVVAQCELHGEEPSPCEFPAWEGEVCGRKVKVSVIEPEENTKLCGPAAFNEVVTYQGDILGIPNTKKWQKAFENHSAMAGIRFIEAFAAQAAREIEEAAMSGADEHIVRVRIVKVPSEVNIKIGATAQRYITGKNKKIDMRGPIFTSAKAEFE *Utilizes base truncation mutantreported in reference (3) with an additional mutation of our own design.**Truncated version of sulfotyrosine tRNA synthetase mutant from (2).The full length mutant is under patent - U.S. Pat. No. 8,114,652 B2.

TABLE B Edmanase Sequence SEQ IDAPAAVDWRARGAVTAVKDSGQCGSGWAFAAIGNVECQWFLAGHPL NO: 29TNLSEQMLVSCDKTDSGCSSGLMDNAFEWIVQENNGAVYTEDSYPYASATGISPPCTTSGHTVGATITGHVELPQDEAQIAAWLAVNGPVAVCVDASSWMTYTGGVMTSCVSESYDHGVLLVGYNDSHKVPYWIIKNSWTTQWGEEGYIRIAKGSNQCLVKEEASSAVVG

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1. An isolated, synthetic, or recombinant N-terminal amino acid bindingprotein (NAAB) comprising: (1) an amino acid sequence having a glutamateresidue at a position corresponding to position 42 of wild-type E. colimethionine aminopeptidase (eMAP) (SEQ ID NO: 1), a tryptophan residue ata position corresponding to position 46 of wild-type eMAP, a threonineor serine residue at a position corresponding to position 56 ofwild-type eMAP, an aspartate residue at a position corresponding toposition 57 of wild-type eMAP, a serine residue at a positioncorresponding to position 58 of wild-type eMAP, a leucine residue at aposition corresponding to position 59 of wild-type eMAP, a threonine orserine residue at a position corresponding to position 60 of wild-typeeMAP, a histidine residue at a position corresponding to position 62 ofwild-type eMAP, an asparagine residue at a position corresponding toposition 63 of wild-type eMAP, a isoleucine or valine residue at aposition corresponding to position 65 of wild-type eMAP, an aspartateresidue at a position corresponding to position 66 of wild-type eMAP, aglycine residue at a position corresponding to position 67 of wild-typeeMAP, a histidine residue at a position corresponding to position 68 ofwild-type eMAP, a glycine residue at a position corresponding toposition 69 of wild-type eMAP, a serine or threonine residue at aposition corresponding to position 70 of wild-type eMAP, a valineresidue at a position corresponding to position 81 of wild-type eMAP, anarginine residue at a position corresponding to position 101 ofwild-type eMAP, a histidine residue at a position corresponding toposition 177 of wild-type eMAP, and a serine or threonine residue at aposition corresponding to position 221 of wild-type eMAP or (2) an aminoacid sequence having a serine residue at a position corresponding toposition 13 of wild-type E. coli methionyl-tRNA synthetase (MetRS) (SEQID NO: 5); a leucine residue at a position corresponding to position 260of wild-type E. coli MetRS; a glycine residue at a positioncorresponding to position 296 of wild-type E. coli MetRS; and a leucineresidue at a position corresponding to position 301 of wild-type E. coliMetRS; wherein the remaining amino acid sequence of the NAAB comprises asequence having at least about 80% sequence identity to the amino acidsequence of SEQ ID NO:
 3. 2. The NAAB of claim 1, wherein the remainingamino acid sequence comprises a sequence having at least 85%, at least90%, at least 93%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the amino acid sequence ofwild-type eMAP (SEQ ID NO: 1).
 3. The NAAB of claim 1, wherein the NAABcomprises the amino acid sequence of SEQ ID NO:
 2. 4. (canceled) 5.(canceled)
 6. The isolated, synthetic, or recombinant N-terminal aminoacid binding protein (NAAB) of claim 1 comprising the amino acidsequence having a serine residue at a position corresponding to position13 of wild-type E. coli methionyl-tRNA synthetase (MetRS) (SEQ ID NO:5); a leucine residue at a position corresponding to position 260 ofwild-type E. coli MetRS; a glycine residue at a position correspondingto position 296 of wild-type E. coli MetRS; and a leucine residue at aposition corresponding to position 301 of wild-type E. coli MetRS;wherein the remaining amino acid sequence of the NAAB comprises asequence having at least about 80% sequence identity to the amino acidsequence of SEQ ID NO:
 3. 7. The NAAB of claim 6, wherein the remainingamino acid sequence comprises a sequence having at least 85%, at least90%, at least 93%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the amino acid sequence of SEQID NO:
 3. 8. The NAAB of claim 6, wherein the NAAB comprises the aminoacid sequence of SEQ ID NO:
 4. 9. (canceled)
 10. (canceled)
 11. Anisolated, synthetic, or recombinant N-terminal amino acid bindingprotein (NAAB) comprising: (1) an amino acid sequence having anaspartate residue at a position corresponding to position 100 ofwild-type PheRS from Thermus Thermophilus (SEQ ID NO: 8), an asparagineresidue at a position corresponding to position 142 of wild-type PheRS,a glycine residue at a position corresponding to position 143 ofwild-type PheRS, a valine residue at a position corresponding toposition 152 of wild-type PheRS, a glycine residue at a positioncorresponding to position 153 of wild-type PheRS, a lysine residue at aposition corresponding to position 154 of wild-type PheRS, an aspartateresidue at a position corresponding to position 165 of wild-type PheRS,an alanine residue at a position corresponding to position 205 ofwild-type PheRS, an alanine residue at a position corresponding toposition 212 of wild-type PheRS, a valine residue at a positioncorresponding to position 228 of wild-type PheRS, an asparagine residueat a position corresponding to position 229 of wild-type PheRS, aglutamate residue at a position corresponding to position 230 ofwild-type PheRS, a glycine residue at a position corresponding toposition 231 of wild-type PheRS, an aspartate residue at a positioncorresponding to position 232 of wild-type PheRS, a tyrosine residue ata position corresponding to position 234 of wild-type PheRS, a threonineresidue at a position corresponding to position 257 of wild-type PheRS,a glycine residue at a position corresponding to position 287 ofwild-type PheRS, an asparagine residue at a position corresponding toposition 289 of wild-type PheRS, an aspartate residue at a positioncorresponding to position 303 of wild-type PheRS, an alanine residue ata position corresponding to position 336 of wild-type PheRS, a threonineresidue at a position corresponding to position 338 of wild-type PheRS,and a glycine residue at a position corresponding to position 340 ofwild-type PheRS; wherein the remaining amino acid sequence of the NAABcomprises a sequence having at least about 80% sequence identity to theamino acid sequence of SEQ ID NO: 6 or (2) an amino acid sequence havingan asparagine residue at a position corresponding to position 121 oftruncated wild-type HisRS from E. coli (3-180) (SEQ ID NO: 9) and analanine residue at a position corresponding to position 122 of wild-typeHisRS; wherein the remaining amino acid sequence of the NAAB comprises asequence having at least about 80% sequence identity to the amino acidsequence of SEQ ID NO:
 9. 12. The NAAB of claim 11, wherein theremaining amino acid sequence comprises a sequence having at least 85%,at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:
 6. 13. The NAAB of claim 11, wherein the NAAB comprisesthe amino acid sequence of SEQ ID NO:
 7. 14. (canceled)
 15. (canceled)16. The isolated, synthetic, or recombinant N-terminal amino acidbinding protein (NAAB) of claim 11 comprising the amino acid sequencehaving an asparagine residue at a position corresponding to position 121of truncated wild-type HisRS from E. coli (3-180) (SEQ ID NO: 9) and analanine residue at a position corresponding to position 122 of wild-typeHisRS; wherein the remaining amino acid sequence of the NAAB comprises asequence having at least about 80% sequence identity to the amino acidsequence of SEQ ID NO:
 9. 17. The NAAB of claim 16, wherein theremaining amino acid sequence comprises a sequence having at least 85%,at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:
 9. 18. The NAAB of claim 16, wherein the NAAB comprisesthe amino acid sequence of SEQ ID NO:
 10. 19. (canceled)
 20. (canceled)21. A polypeptide of claim 1, wherein the NAAB is coupled to afluorophore.
 22. The polypeptide of claim 21 wherein the fluorophorecomprises Cy3 or Cy5.
 23. An isolated, synthetic, or recombinant Edmandegradation enzyme comprising an amino acid sequence having a glycineresidue at a position corresponding to position 25 of wild-typeTrypanosoma cruzi cruzian; a serine residue at a position correspondingto position 65; a cysteine residue at a position corresponding toposition 138; and a tryptophan residue at a position corresponding toposition 160; wherein the remaining amino acid sequence of the Edmandegradation enzyme comprises a sequence having at least about 80%sequence identity to the amino acid sequence of wild-type Trypanosomacruzi cruzian.
 24. The Edman degradation enzyme of claim 23, wherein theremaining amino acid sequence comprises a sequence having at least 85%,at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof wild-type Trypanosoma cruzi cruzian.
 25. The Edman degradation enzymeof claim 23, wherein the enzyme comprises the amino acid sequence of SEQID NO:
 29. 26. The Edman degradation enzyme of claim 23, wherein theenzyme consists of the sequence of SEQ ID NO:
 29. 27-48. (canceled) 49.A kit for sequencing polypeptides comprising: one or more N-terminalamino acid binding proteins (NAABs); the Edman degradation enzyme ofclaim 23; and instructions for using the NAABs and the Edman degradationenzyme for sequencing a polypeptide.
 50. (canceled)
 51. The kit of claim49 wherein the NAAB comprises one or more amino acid sequences selectedfrom the group consisting of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 7;SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO:14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ IDNO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; and SEQ ID NO:28. 52-83. (canceled)